Gaza has been under an Israeli blockade since 2006 but it was tightened in 2007 after the Islamist Palestinian group Hamas seized control in the tiny enclave.
Israel says its maritime, land and sea blockade of Gaza is aimed at preventing Hamas from receiving supplies which could be used for military purposes.
Fifteen women will try to breach the blockade aboard the Zaytouna-Oliva boat early on Wednesday, said spokeswoman Claude Leotic.
"But we fear there will be an Israeli attack" to prevent the boat from reaching Gaza's shores, she told AFP Tuesday in a telephone interview.
Israeli media, quoting unnamed officials, have reported in recent days that the navy will intercept the boat and escort it to the Israeli port of Ashdod to prevent it from reaching Gaza.
The Zaytouna-Oliva is one of two vessels that set sail from Barcelona in September.
Signup to our newsletter and follow us on Facebook and Twitter!
The flotilla dubbed "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 to 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.
Among the women of different nationalities on board the Zaytouna-Oliva is Northern Ireland activist and 1976 Nobel Peace Prize winner Mairead Maguire.
A South African passenger, Leigh-Ann Naidoo, told AFP she expected the boat to be 100 nautical miles off the shores of Gaza by 0300 GMT.
"Our goal is to reach Gaza. We are not worried about what Israel plans on doing," she added.
The Gaza blockade also severely restricts the movement of 1.9 million Palestinians who live in the impoverished territory.