Tripoli had been a problem for the Knights Hospitaller since Spain handed it to them in 1530, bundled together with Malta and Gozo as a consolation prize for losing Rhodes. The city had been under Spanish rule since its capture in 1510, and the local population, displaced and resentful, had never accepted Christian governance. Nearby Tajura, whose inhabitants included descendants of those driven out by the Spanish invasion, maintained open hostility. The fortifications were modest: packed earth walls, a partly stone fortress facing the sea, and a smaller stronghold called the castillegio guarding the harbor mouth. In the 1540s, when Jean Parisot de Valette served as Governor of Tripoli, the Order briefly considered moving its headquarters from Malta to the North African outpost. Ottoman Sultan Suleiman the Magnificent may have been motivated to take Tripoli precisely to prevent such a consolidation of Hospitaller power.

Before reaching Tripoli, the Ottoman fleet struck Malta's sister island of Gozo, invading and sacking it. Between 5,000 and 7,000 of Gozo's inhabitants were seized and enslaved, a devastating blow that emptied entire communities. Led by the admiral Sinan Pasha and the legendary corsair Dragut, the fleet then sailed south to the North African coast. At Tajura, the local population under Murad Agha eagerly joined the Ottoman force, their long-simmering grievances against the Hospitallers finding a powerful ally. Ottoman troops landed at both Zuwarah, west of Tripoli, and Tajura to its east, encircling the city.

An unlikely figure appeared in the middle of the crisis: Gabriel d'Aramont, the French ambassador to the Ottoman Empire, who arrived in Malta shortly after the attack on Gozo. The Hospitallers persuaded d'Aramont and his secretary, Nicolas de Nicolay, to sail to Tripoli and attempt to negotiate. They departed Malta on August 2 with three ships, but by the time they reached the city, Ottoman forces were already positioned in Tajura. Thanks to the Franco-Ottoman alliance, the Ottoman fleet saluted the French envoys and permitted them to land. Governor de Vallier, facing overwhelming force with meager defenses, capitulated after six days of bombardment. D'Aramont's intervention secured safe passage for the Hospitaller knights and part of the garrison to return to Malta. The rest, including Maltese and Rhodian soldiers and Calabrian mercenaries who had mutinied during the siege, were not so fortunate. They were massacred or enslaved.

After the fall of the city, Sinan Pasha appointed Murad Agha as beylerbey of Tripoli. Murad Agha strengthened the fortifications and built a fortified mosque at Tajura, consolidating Ottoman control over the region. But the corsair Dragut, who had been promised the governorship before joining the expedition, left North Africa in protest and sailed to Constantinople, where the fleet's crews declared they would serve under no other commander. Suleiman placated Dragut by offering him the governorship of Karlieli and instructing Sinan Pasha to defer to his orders. Dragut would eventually succeed Murad Agha and transform Tripoli into one of the Mediterranean's most formidable corsair bases, a role the city would play for centuries under Ottoman rule.

The recriminations were swift and wide-ranging. Ambassador d'Aramont was criticized by Holy Roman Emperor Charles V and Pope Julius III, who suspected he had encouraged the Ottomans to attack. The Calabrian soldiers, enslaved and unable to defend themselves, were conveniently blamed for the city's fall. On September 11, 1551, the Pope urged Grand Master Homedes to relocate the Order's headquarters to Syracuse or Messina in Sicily. The Hospitallers began plotting to retake Tripoli almost immediately, raiding its hinterland in 1552, but the city would never return to Christian rule. Villegaignon and Nicolay published accounts of the siege within a few years, leaving behind firsthand records of the battle that ended the Hospitaller presence on the African coast and opened a new chapter in Tripoli's long history as a contested crossroads of the Mediterranean.