"We think that through this act organised by women, we can give more visibility to the important role of Palestinian women in the fight for freedom," said Zohar Chamberlain, one of the organisers, just before two yachts left the Spanish Mediterranean city.
Chamberlain, an Israeli living in Spain, told AFP she felt she had a "double duty" to denounce the land, sea and air blockade imposed in 2006 on Gaza, which is controlled by Islamist group Hamas.
It "doesn't only make Palestinians suffer, it corrupts the souls of Israelis as one can't remain human if one treats our Palestinian brothers as if they weren't our brothers."
Israel says the blockade is necessary to prevent Hamas from receiving materials that could be used for military purposes.
But the World Bank and United Nations say it has put an end to virtually all exports from Gaza and undermined the economy of this small coastal enclave stuck between Egypt, Israel and the Mediterranean.
The blockade also severely restricts the movement of 1.9 million Palestinians living in Gaza.
The flotilla that set sail from Barcelona, called "Women's Boat to Gaza", is part of the wider Freedom Flotilla Coalition that consists of pro-Palestinian boats that regularly go to Gaza from all over the world to try and break the blockade.
None have yet managed to get through, and Israeli authorities have made several arrests.
One such operation turned to tragedy in 2010 when Israeli commandos killed 10 Turkish activists in a raid on a flotilla.
The women travelling on board the two boats come from countries such as the United States, Norway, South Africa or Malaysia.
They are due to stop in several ports on the way, including Ajaccio in France's Corsica, to take other activists on board and hope to arrive in Gaza in early October.