The Global Sumud Flotilla Mission, carrying aid and pro-Palestinian activists, including climate campaigner Greta Thunberg, set sail for Gaza on Monday.
This comes after the aid flotilla of dozens of boats temporarily returned to port in Barcelona due to stormy weather, organizers said.
"We conducted a sea trial and then returned to port to allow the storm to pass. This meant delaying our departure to avoid risking complications with the smaller boats," the Global Sumud Flotilla Mission said in a statement, adding winds had been up to around 35 mph (56 kph).
Activists, including Game of Thrones actor Liam Cunningham, are on the flotilla of several dozen boats that is aiming to break Israel's naval blockade and deliver food and humanitarian supplies to the enclave, which has been devastated by nearly two years of war.
Israel has argued that the naval blockade it imposed in 2007 is necessary to stop weapons from being smuggled to the Hamas Palestinian militant group. It has described other attempts to break it - including one that included Thunberg in June - as a propaganda stunt in support of Hamas.
Previous 'Freedom Flotilla' attempts
Two previous attempts to sail to Gaza were made by activists during July, with both finally stopped by the IDF when they approached Israel's shores.
Thumberg in particular sailed with the Madleen, a sailboat that was seized by the IDF while its passengers were detained and later deported back to their countries.
The second attempt, done with the Handala ship, was also intercepted by the Israeli Navy, and the activists aboard were also deported.