Two years earlier, in 1462, Mehmed the Conqueror had taken Lesbos and ended a century of Genoese rule. Venice could not let the island rest in Ottoman hands. The Republic worried that the sultan might trade it to the Florentines, their commercial rivals, and Lesbos sat astride the sea lanes Venice depended on. So in 1464, as the First Ottoman-Venetian War spread across the Aegean, the Venetian senate authorized Giustinian to attack. There were other tempting targets, fortresses guarding the Dardanelles among them, but Lesbos remained the prize. The admiral assembled a powerful force and pointed it at the same harbor city that had defied Rome and then Mehmed.

The fleet was formidable: by one account 32 galleys, four ships carrying 800 infantry, and heavy artillery; by another, seventy ships and three thousand heavy infantry. Against them stood roughly 400 of the sultan's heavily armored guards, almost certainly Janissaries, behind the walls of Mytilene. The first assault came on April 1. The Venetians brought stone-hurling cannon, crossbows, and scaling ladders. They called on the garrison to surrender. The garrison refused. The bombardment opened only a small breach, and the defenders rebuilt it. The Venetian cannon began to crack and burst after repeated firing. Assault after assault, by mine and by ladder, broke against the defense, and the casualties mounted on the Venetian side with nothing to show for them.

What finally ended the siege was not a breach but news. Mehmed dispatched a relief fleet of 150 vessels under Mahmud Pasha Angelović, the same commander who had taken the island in 1462. When word reached the Venetians that this armada was coming, they did not wait to be trapped against the shore. On May 18 they broke off the siege in haste, leaving their artillery behind and carrying away the islanders who had thrown in their lot with them. Giustinian withdrew to Negroponte, the great Venetian base on Euboea, and gathered what was left of his pride for one more attempt.

The second try was a raid by Stratioti, Balkan light cavalry, and galley crews. It failed as completely as the first. Giustinian sailed on to Methoni and died there in July, undone, the sources insist, by the bitterness of his defeat. The siege left no monument, only a number: five thousand dead beneath the same walls that had repelled so many before. For Lesbos, the consequence was permanence. Venice would not seriously threaten the island again, and Mytilene settled into four and a half centuries of Ottoman rule. The castle that turned back the Venetian guns still stands above the harbor, weathered and patient, a fortress that outlasted everyone who tried to take it by force.