An all-woman Gaza-bound boat will aim to break the Israeli blockade on Wednesday.
But the Israeli navy is preparing to intercept the aid vessel, which hopes to breach the decade-long blockade on the Palestinian enclave.
The group of 15 activists set sail from Barcelona three weeks ago and hopes to reach Gaza's shores in the morning, spokeswoman Claude Leotic told AFP.
"But we fear there will be an Israeli attack," she added.
Gaza has been under siege since 2006 but it was tightened in 2007 after the Hamas took control of the strip.
Israel says its maritime, land and sea blockade of Gaza is necessary to prevent Hamas receiving supplies which could be used for military purposes.
But the World Bank and United Nations said it has put an end to virtually all exports from Gaza and undermined the economy of the small coastal enclave.
The blockade also severely restricts the movement of the 1.9 million Palestinians living in the impoverished territory.
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.
Two boats left Barcelona on 14 September - Amal and Zaytona. Amal was anchored on Monday due to engine problems, but Zaytouna has continued to Gaza.
On Sunday, the Israeli navy received instructions to intercept the boat and arrest everyone on board, Israeli daily newspaper Maariv has reported.
Israeli news website breakingisraelnews.com said officers had been preparing to "exercise the routine protocol in such cases, namely to board the boats and escort the passengers, politely but firmly".
"Our goal is to reach Gaza. We are not worried about what Israel plans on doing," said South African activist Leigh-Ann Naidoo.
A similar operation turned to tragedy in 2010 when Israeli commandos killed 10 Turkish activists in a raid on a flotilla.