The end of the Medieval period on the Maltese Islands saw the arrival of the Order of St. John of Jerusalem, an order with strong hospitallier traditions. The arrival of the Order served to maintain and introduce medical concepts which developed in Europe during the sixteenth and subsequent centuries. The Order of the Knights Hospitaliers of St. John had its origins in Jerusalem in the early years of the first millennium AD, when the Benedictines with the help of some merchants built hospitals to cater for the needs of pilgrims. Increasing harassment by the Turks necessitated changes in the organization and functions of the Order. Thus to the religious and nursing duties were added the chivalrous ones of defending pilgrims to the Holy Land. They were ousted from Jerusalem in 1187, and were progressively pushed westwards by the Turks, until finally they were ousted from Rhodes in the beginning of the sixteenth century. The Maltese Islands were ceded to them in 1530 by Emperor Charles V of Spain. They continued to rule for just under three centuries influencing the Islands until the end of the eighteenth century, when they were expelled from the Islands by Napoleon Bonaparte in 1798. The French influence was short-lived since the Maltese rebelled and blockaded the French in the towns with the help of the British. The French commanders capitulated on the 5th September 1800.

The Military Knights were the sons of the great houses of France, Spain, Italy, Germany, and England. The French influence predominated. This cosmopolitan group enabled the Islands to maintain a strong cultural attachment to the mainland in Europe and thus benefit from the renaissance movement on the continent. This cultural movement, characterized by the re-awakening of ancient learning through direct knowledge of Greek and Roman authors, was not confined to the arts. It resulted in a new general outlook with emphasis on knowledge of nature and the view of man as nature's masterpiece. This was the beginning of Humanism. Interest in classical antiquity was cultivated, while metaphysical speculations ceased and the fetters on learning imposed by the medieval attitude towards religion fell away. Observation of phenomena replaced theoretical procedures. Science advanced from the dark into new territories. The same held true for medicine.

The arrival of the Order to the Islands brought an influx of physicians and surgeons to the islands, thus correcting the deficiency of medical personnel experienced in Malta during the late Middle Ages resulting from the expulsion of the Jews from the Islands. At Rhodes, the Knights had a code of laws designed to safeguard the health of the community. These laws were comprehensive and dealt with measures to control infectious disease, the issuing of licenses to practice medicine and surgery, and to identify the responsibilities of apothecaries and medical men. This code of laws was introduced in the Islands, and was amended and modified throughout the subsequent centuries by a number of Protomedical decrees until the publication of the legal code of 1724 which was eventually incorporated in the De Rohan Code of 1784. The Church authorities further exercised control particularly in the practice of midwifery and treatment using witchcraft. This control was enforced either by the requirement of licenses to practice midwifery or by the vigilance of the Inquisition.

In the early years of the sixteenth century, the Order was primarily concerned with the defense of the Islands against the Turkish gradual invasion of the Mediterranean. The Knights concentrated their forces in the maritime center of Birgu. There they established their first hospital. In 1532, after expropriating and demolishing as number of buildings on the foreshore of the town, the building of the Holy Infirmary was started. Besides catering for members of the Order, the hospital was also open to male civilians while it also cared for orphans and foundlings. A few years after its completion, the Infirmary became incapable of providing sufficient beds, and the hospital was enlarged by the addition of another storey. While this extension solved the day-to-day requirements, hospital accommodation still fell short in times of emergency. The Birgu Infirmary functioned until 1575 when the new Sarca Infirmeria was build in Valletta. At Birgu, the Italian Knights also kept a small hospital, built around 1554, in their own auberge.

Following the Knights victory over the Turks in 1565, the Order decided to build a new fortified city guarding the major harbour of the Islands. A new Infirmary was planned, the site chosen being the south-eastern side of the new capital city. The building, started in 1574, consisted originally of one long ward. It was subsequently enlarged in 1583 by the addition of a new block, and remodeled in 1662 and in the eighteenth century. The hospital wards were open for member of the order, civilians and slaves. The Valletta Holy Infirmary was one of the best serviced hospitals in Europe and was favourably described by a number of foreign visitors to the Islands during the seventeenth and eighteenth centuries. The hospital was converted into a military hospital during the late eighteenth century under the French and continued to be used thus during the nineteenth century by the British. The Sacra Infirmeria mainly served male patients, though it also catered for foundlings. In 1625 Caterina Scarpi set apart a small house - the Santa Maria della Scala - in Vallettaa for the care of poor infirm women. This small woman's hospital was closed down after its foundress died in 1655, but in 1659 a new women's hospital - the Casetta - was re-established by Grandmaster de Redin. Other general hospitals were established in Gozo.

Medical practice at the beginning of the modern period was primarily based on the teachings of ancient and medieval authors including the works of Galen, Rhazes, Avenzoar and Avicenna. The physicians of the Order kept well abreast with medical developments on the Continent. A number of these in the 17th and 18th centuries are known to have proceeded abroad to Italy and France to further their studies abroad. Other Maltese physicians such as Mikel'Angelo Grima published medical papers locally and abroad. The medicines available to sixteenth century physicians generally included vegetable sources, and a minority of animal and mineral sources. The arrival of the Knights appears to have increased the number of substances available through a greater proportion of inported substances. These medicaments, some of which were completely useless, appear to have been retained well into the eighteenth century.

Mikel'Angelo Grima epitomizes the pattern of training received by Maltese practitioners in the eighteenth century. Grima, born in Valletta in 1731, started his surgical training at the age of twelve years at the Sacra Infirmeria. In 1750 he left for Florence where he joined the medical school of Santa Maria Nuovo. He obtained his doctorate in philosophy and medicine from the University of Pisa in 1754, but continued to work in Florence where he conducted experiments on methods of intestinal surgery. In 1758 he was granted a subsidy by the Order to continue his studies in Paris, later joined the French forces as surgeon during the Seven Years War. In 1763, Grima was recalled back to Malta to begin his duties in the medical service of the Order. He was appointed Chief Surgeon at the Sacra Infirmeria and eventually in 1765 was appointed to the Chair of Anatomy and Surgery. He died in 1798.

Medical teaching in the early part of the Renaissance remained, on the surface, like that of the Middle Ages with many teachers continuing to express the conservative views of Galen and Avicenna. Nevertheless, new attitudes slowly became evident particularly towards the acquisition of anatomical knowledge. Public dissection was practiced in ever-increasing numbers, and towards the end of the sixteenth century anatomy theaters began to be built in various European universities. Anatomy, the study of which is necessary for the rational approach to medicine, was to be reborn in the sixteenth century with the works of Leonardo da Vinci and Andreas Vesalius. There was during this period an integration between art and anatomy. This integration was also evident in Malta. The celebrated French artist Antoine Favray who lived in Malta in the latter half of the eighteenth century serving with the Order studied the skeletal and muscular movements of the human body and prepared anatomical drawings intended for the use of students in the medical school. The School of Anatomy and Surgery in Malta was established in 1676 by Grandmaster Fra Nicolas Cottoner with the first teacher being Dr. Fra Giuseppe Zammit. Throughout the seventeenth and eighteenth centuries, there was a marked progress in the teaching of Anatomy in Malta, though with occasional setbacks. The School of Anatomy in the eighteenth century came to acquire great renown throughout the principal cities of Europe, particularly under the directorship of Dr. Gabriele Henin and Mikel'Angelo Grima. Henin has been regarded the Father of Anatomy in Malta. Though primarily a surgeon, Henin was in 1721 sent to Florence to learn practical anatomy. He was recalled back to Malta when the Chair of Anatomy became vacant in 1723. He lectured on Anatomy and introduced classes of dissection and demonstrations on the human cadaver in public, besides performing all postmortem examinations. His death in 1754 was setback to the School, until Mikel'Angelo Grima was appointed in 1763. The School of Anatomy was the prelude to the Faculty of Medicine at the University of Malta. Following the expulsion of the Jesuits from Malta in 1769, the Society's property including their college erected in Valletta for the education of young men was taken over by the Order. Out of the revenues accruing from this property, a university was founded by Grandmaster Em. Pinto de Fonceca. The three faculties of Theology, Law and medicine were established in 1771 and the Chair of Anatomy and Surgery were incorporated in the new institution.

In spite of progress in medicine and improvements in social conditions, seventeenth century Europe suffered many epidemics as bad or worse as those of the Middle Ages. The Maltese islands in the Central Mediterranean and an important maritime base likewise was subject to the repeat introduction of various epidemic infectious disease. Thus plague or the Black death was introduced to the Islands in 1501, 1519, 1523, 1575, 1592, 1623, 1655, and 1675; smallpox in 1680, 1763, 1769, and 1780; while cholera was introduced in 1745, 1767, 1770, and 1783. Quarantine measures had long been introduced in the Mediterranean in attempts to control the spread of infectious disease. Under the Order of St. John, the quarantine measures were re-enforced. All merchandise was brought ashore at the Lazaretto on Manoel Island for disinfection. As paper was considered a potential vehicle of contagion, letters from abroad were disinfected by dipping in vinegar or exposure to the fumes of burning straw and a mixture of manganese and sulphuric acid. Passengers were detained in the quarantine establishment for up to forty days and exposed to "smoking" from the burning of aromatic herbs before allowed freedom of communication with the inhabitants. Infected ships were sometimes towed out to sea and destroyed with their cargo. The same quarantine measures were retained well into the nineteenth century under British rule.

The end of the middle Ages in the sixteenth century heralded the onset of the renaissance and the beginning of Humanism. The seventeenth century can be referred to as the "Golden Age of Science". However during this century the gulf between medical practice and advances in research was wider than at any one time before or since. The theory and practice of medicine remained in the Arabo-Hellenic tradition well into the eighteenth century. Medical practitioners were for the most part poorly trained and loath to keep abreast of developments, continuing to prescribe the same old remedies of enemas, blood-letting and purging whatever the disease. This century presented a paradoxical situation in which the natural sciences were developing broadly and swiftly but medicine seemed to be if anything retreating into a more dogmatic attitude that it had adopted during the Renaissance. Alchemy and the sale of panaceas flourished, while surgery seemed to benefit little from the great advances of the previous century. By the mid-seventeenth century, the efforts to free medical thought from the doctrines of the ancients was almost complete. The situation became fluid with many new concepts more or less tenable. Medicine could now advance and the discipline of the exact sciences created an environment for future victories over disease. The eighteenth century was the "Age of Enlightenment" where besides looking increasingly inwards, physicians came more and more to accept only what was observable directly and reproducible by experiment. This attitude set the pattern of scientific thought for the nineteenth century. The constant traffic between the Maltese Islands and the European continent, particularly Italy and France, resulted in practitioners practicing on the Islands following the same concepts and trends as on the continent, sometimes contribution actively in the process of medical advancement. The end of the eighteenth century in Malta was marked with a period of strife after the Islands were taken over by Bonaparte in 1798. The Maltese rebelled and blockaded the French in the towns for two years. These two years were attendant with a general disruption of the way of life for both the general populace and the besieged French with the accompaniment of disease and famine. The political changes at the turn of the eighteenth century paved the way for a change in direction with regards to medical education.