While based in Malta, the Order and its navy participated in a number of naval battles against the Ottoman Navy or the Barbary pirates. The Order sent a carrack and four galleys to support the Spanish Empire and its allies in the conquest of Tunis in 1535. It also participated in the Battle of Preveza (1538), the Algiers expedition (1541) and Battle of Djerba (1560), in which the Ottomans were victorious over the Christian forces.

Four of the Order's galleys, Santa Fè, San Michele, San Filippo and San Claudio, capsized in a tornado in the Grand Harbour in 1555. They were replaced with funds sent from Spain, the Papal States, France and the Prior of St. Giles. One galley was built at the expense of Grand Master Claude de la Sengle.

When the city of Valletta began to be built in the 1560s, there were plans to build an arsenal and mandracchio for the Order's navy. The arsenal was never built, and while work started on the mandracchio, it stopped and the area became a slum known as the Manderaggio. Eventually, an arsenal was built in Birgu in 1597. A dock was built in Valletta's ditch in 1654, but it closed in 1685.