On Rhodes the Hospitallers, by then also referred to as the Knights of Rhodes, were forced to become a more militarized force, fighting especially with the Barbary pirates. They withstood two invasions in the 15th century, one by the Sultan of Egypt in 1444 and another by the Ottoman Sultan Mehmed the Conqueror in 1480 who, after capturing Constantinople and defeating the Byzantine Empire in 1453, made the Knights a priority target.

In 1522, an entirely new sort of force arrived: 400 ships under the command of Sultan Suleiman the Magnificent delivered 100,000 men to the island (200,000 in other sources). Against this force the Knights, under Grand Master Philippe Villiers de L'Isle-Adam, had about 7,000 men-at-arms and their fortifications. The siege lasted six months, at the end of which the surviving defeated Hospitallers were allowed to withdraw to Sicily. Despite the defeat, both Christians and Muslims seem to have regarded the conduct of Phillipe Villiers de L'Isle-Adam as extremely valiant, and the Grand Master was proclaimed a Defender of the Faith by Pope Adrian VI.