"Israel should not be under any illusion whatsoever that their threats or intimidation will stop us or even that their violence against us will stop us," Huwaida Arraf, from the Free Gaza Movement, said.
Kahel Mazen, from EGESG, added: "If they [the Israelis] choose to resort to destruction and death, our mission is to try to open a window of hope for the people of Gaza."
A statement from Israel's foreign ministry said it had "no intention of allowing the flotilla into Gaza" but has not elaborated on what measures could be used to stop them.
The flotilla will be made up of three cargo ships and five passengers boats.
One cargo ship, the MV Rachel Corrie, named after a US human rights activist killed by an Israeli bulldozer, set sail from Ireland last week.
The remainder are expected to leave by May 23 from a range of destinations including Turkey and Greece.
Greta Berlin, one of the flotilla's organisers, told Al Jazeera that despite being "brutally attacked" for attempting to enter Gaza in the past, the movement was optimistic about the latest attempt.
"The last three trips we have gone on we were brutally attacked," she said.
"That was only with one boat, this time we have three cargo ships and five passenger boats.
"We're going into the waters of Gaza, not Israel, so they have no right to stop us."
She said a number of high-profile people would be on board the boats, including members of parliament from several participating countries.
Hundreds of activists protesting against Israel's siege on Gaza will also be aboard the vessels.
Israel says the blockade aims to prevent Hamas, the political movement that controls Gaza, from acquiring weapons or materials that could be used for military purposes.
For the majority of Gaza's population of 1.5 million people, the result has been impoverished living conditions, while Hamas' grip on power since 2007 shows little sign of weakening.