The castle sat on a narrow spit of land at Lefkada's northeastern tip, protected by marshes to the south and by a wide moat on the other sides. Access was only possible over wooden bridges at the western and eastern gates. A 3-kilometre aqueduct, built in 1564, ran along a footpath that was otherwise the island's only land connection to the interior. The fortress itself, rebuilt after the Ottoman defeat at Lepanto in 1571, was an irregular hexagon some 220 metres by 150 metres, with nine large round cannon bastions. Inside lived a walled community: according to the traveller Evliya Celebi, who had visited just over a decade earlier, around 200 stone houses occupied by Muslims, with wooden suburban settlements of Christians outside the walls, and a further suburb on the island itself. The garrison in 1684 numbered 500 Albanians and 200 Greeks.

Morosini's fleet of 38 galleys, 8 galleasses, and 22 sailing men-of-war arrived on 20 July. The assault was designed to strike simultaneously from the mainland to the east and the island to the west. On 21 July, the Ottoman commander Bekir Agha rejected terms for surrender. Greek auxiliary forces, including 150 fighters under the pirate captain Stathis Manetas and 2,000 Greeks from the Venetian-held Ionian Islands under Colonel Giovanni-Battista Metaxa, joined the Venetian lines. The fleet's naval bombardment was hampered by contrary winds; one galley was nearly sunk. Manetas and his men worked at night, throwing burning material over the walls to ignite the timber buildings inside. A bastion was severely damaged on 27 July and collapsed on the 31st. By that point the garrison was running out of ammunition. Morosini was meanwhile concerned about a possible relief force from the Ottoman garrison at Preveza; on 2 August the fleet bombarded the Castle of Bouka to forestall any intervention. On 6 August the Venetians launched their main assault on the breach — and were driven back, with 40 soldiers killed and many more wounded. That same evening, Bekir Agha offered to surrender on terms.

The garrison and approximately 3,000 civilian residents left the fortress the following day, with safe passage guaranteed. They left behind 126 pieces of artillery. The Venetian losses in combat were 127 dead and 128 wounded. Disease took a heavier toll: 1,740 soldiers had to be evacuated to Corfu for treatment, a number that indicates the miserable conditions of a siege in summer heat. Among those freed when the fortress fell were 137 people held as Christian slaves; under Morosini's terms they were compelled to serve a year in the Venetian fleet or army before receiving their freedom. Of 42 enslaved Black men, some were sent to the galleys and the others distributed among the officers — an act that the source records without commentary, and which we note here as what it was: the continuation of slavery under a different master. The walled town and its two outer suburbs were then razed to create a clear glacis around the fortress. The community that had lived inside those walls for generations was erased.

The fall of Santa Maura opened the Morean War, a conflict in which the Republic of Venice would spend the next fifteen years recovering territory it had lost to the Ottomans across Greece and the Adriatic. Morosini went on to lead campaigns in the Peloponnese, earning the nickname "the Peloponnesian" and eventually becoming Doge of Venice. The fortress at Lefkada was modernised over the following decades, with strengthened ramparts on the eastern side facing the Ottoman-held mainland. In 1715, during the Seventh Ottoman-Venetian War, the Venetians briefly abandoned it rather than overextend their forces — but recaptured it after their victory at the Siege of Corfu. The castle remained a military installation, passing through French, Russian, and British hands before Greek forces garrisoned it after 1864. It was in use until 1922. The modern city of Lefkada stands on the suburb that survived August 1684.