The voyage is organized by the activist group Global Sumuda Flotilla, which calls itself an independent organization, denying any affiliation with any country or political party.
Two previous attempts to deliver aid to the Gaza Strip, in June and July, failed. Israel blocked the activists' boats and detained them, but later expelled them.
Activists are demanding the opening of a humanitarian sea corridor to deliver food, drinking water and medicine to the Gaza Strip.
About 20 boats and activists from 44 countries set off, organizers of the initiative said, adding that it was the largest attempt to break the blockade of the Gaza Strip to date. Hundreds of people, including actors Liam Cunningham, Edward Fernandez and Susan Sarandon, as well as several European politicians and public figures, including the former mayor of Barcelona, Ada Kula, have set off.
They are expected to be joined by other boats from Italy, Greece and Tunisia in the coming days, organizers said. They predict the boats could reach the Gaza Strip in mid-September.
The Israeli army declared Gaza City a"dangerous combat zone" on Friday ahead of a planned offensive to capture the Gaza Strip's largest city. On Saturday, an Israeli official said Israel would soon halt or slow humanitarian aid deliveries to the northern Gaza Strip as it expands its military offensive against the Palestinian militant group Hamas.