The Venetian cartographer Vincenzo Coronelli reports that Spinalonga was not always an island, but was once linked with the adjacent Peninsula Spinalonga. He mentions that in 1526, the Venetians cut down a portion of the peninsula and thus created the island. Because of its position the island was fortified from its earliest years in order to protect the entranceway of the port of Ancient Olous.
In 1578 the Venetians charged the engineer Genese Bressani to plan the island’s fortifications. He created blockhouses at the highest points of the northern and southern side of the island, as well as a fortification ring along the coast that closed out any hostile disembarkation. In 1579, the Provveditore Generale of Crete, Luca Michiel, put the foundation stone of the fortifications, built over the ruins of an acropolis. There are two inscriptions that cite this event, one on the transom of the main gate of the castle and the other on the base of the rampart at the north side of the castle. In 1584, the Venetians, realising that the coastal fortifications were easy to conquer by the enemies attacking from the nearby hills, decided to strengthen their defence by constructing new fortifications at the top of the hill. The Venetian fire would thus have bigger range, rendering Spinalonga an impregnable sea fortress, one of the most important in the Mediterranean basin.
Spinalonga, along with Gramvousa and Souda, remained in Venetian hands even after the rest of Crete fell to the Ottomans in the Cretan War (1645–1669) and until 1715, when they fell to the Ottomans during the last Ottoman – Venetian War. These three forts defended Venetian trade routes and were also useful bases in the event of a new Venetian-Turkish war for Crete.
In 1715 , the Ottoman Turks captured Spinalonga taking over the last remaining Venetian fortress and removing the last trace of Venetian military presence from the island of Crete.
The island was subsequently used as a leper colony from 1903 to 1957. It is notable for being one of the last active leper colonies in Europe. The last inhabitant, a priest, left the island in 1962. This was to maintain the religious tradition of the Greek Orthodox church, in which a buried person has to be commemorated at following intervals of 40 days; 6 months; 1 year; 3 years; and 5 years, after their death. Other leper colonies that have survived Spinalonga include Tichileşti in Eastern Romania, Fontilles in Spain and Talsi in Latvia. As of 2002, few lazarettos remain in Europe.
There were two entrances to Spinalonga, one being the lepers’ entrance, a tunnel known as «Dante’s Gate». This was so named because the patients did not know what was going to happen to them once they arrived. However, once on the island they received food, water, medical attention and social security payments. Previously, such amenities had been unavailable to Crete’s leprosy patients, as they mostly lived in the area’s caves, away from civilization.