Thousands of supporters gathered at Barcelona's port to see off the boats, many of them waving Palestinian flags and chanting: "Free Palestine" and "It's not a war, it's a genocide".
"This is a mission to challenge the extremely violent, business-as-usual international system that is failing to uphold international law," Thunberg told the crowd before the departure of the flotilla of dozens of boats, set to be joined by more along the way.
The Swedish campaigner tried unsuccessfully to break Israel's longstanding naval blockade of Gaza by sailing to the territory in June with other activists.
Israeli forces seized their small aid ship and they were deported from Israel.
Israel has argued that the blockade imposed in 2007 is necessary to stop weapons being smuggled to the Hamas militant group and has described other attempts to break it - including Thunberg's in June - as a propaganda stunt in support of Hamas.
Organisers of the flotilla blamed global leaders for failing to put pressure on Israel to allow aid to pass after a global hunger monitor said part of Gaza was suffering from famine.
The flotilla will be joined by more boats setting off from Greece, Italy and Tunisia, said Yasemin Acar, a member of the steering committee.
In the north-western Italian port of Genoa, some 250 metric tonnes of food for Gaza have been collected from local groups and residents, organisers said.
Some of the aid was loaded on board boats setting off from Genoa on Sunday, while the rest will be sent to the Sicilian port of Catania, from where more vessels are due to leave for Gaza on September 4.
The blockade has remained in place through conflicts, including the current war, which began when Hamas-led militants rampaged through southern Israel on October 7, 2023, killing more than 1200 people and taking 251 hostages, according to Israeli tallies.
Israel's military campaign in Gaza has killed more than 63,000 people, mostly civilians, according to Gaza health officials, and it has plunged the enclave into a humanitarian crisis and left much of it in ruins.
