The activists, Palestinian-Spanish citizen Saif Abukeshek and Brazilian citizen Thiago Avila, were among dozens of activists intercepted by the Israeli navy off the coast of Crete.
They are members the Global Sumud Flotilla's steering committee, whose mission was to break Israel's naval blockade and bring humanitarian aid to the Palestinian territory.
Some 20 boats and 175 activists were intercepted by the Israeli navy.
Six Australians were among those detained, from a total of 17 who were known to be volunteering on the flotilla.
Activists said Israeli forces stormed their vessels, smashed engines and detained some of those onboard.
The incident occurred hundreds of kilometres from Gaza and Israel overnight from Wednesday to Thursday.
Israeli officials said they needed to take early action against the flotilla before it reached Israeli waters because of the high number of boats involved.
The Israeli foreign ministry said on X that it was taking the two activists to Israel for questioning, and that Abukeshek was "suspected of affiliation with a terrorist organisation" and Avila was "suspected of illegal activity", without providing evidence.
The Global Sumud Flotilla appealed for international support.
"We demand that all governments do all they can to pressure the Israeli regime to release all the illegal abductees," the group said on Friday.
The rest of the flotilla participants were released in Crete late on Thursday.
Of the 53 vessels that had been sailing before the interception, 31 reached safe waters and would continue their attempts to "break the illegal siege of Gaza", organisers said.
The flotilla set sail earlier in April from Barcelona, Spain.
The Greek foreign ministry said it had asked Israel to withdraw its ships from the area and had offered its "good services" for the activists to disembark in Greece and be repatriated.
Protests in solidarity with the flotilla erupted across several capitals including in Rome, Athens and Istanbul.
Spain and Brazil have not yet commented on the detention and transfer to Israel of Abukeshek and Avila.
But they said in a joint statement with several other nations that Israel's interception of the flotilla and detention of the activists in international waters were flagrant violations of international law.
The flotilla's latest attempt to reach Gaza comes less than a year after Israeli authorities foiled a previous effort by the group.
That attempt involved about 50 vessels and around 500 activists, including Swedish climate activist Greta Thunberg, Nelson Mandela's grandson Mandla Mandela, and several MPs.
Israel arrested, detained and later deported the participants, including Avila, who claimed Israeli authorities abused them while in detention, which Israel denied.
