Two Spanish activists who were on board the aid flotilla raided by Israeli troops on May 31 on Friday sued Israel for war crimes and crimes against humanity.
Nine Turkish activists were killed in the attack, DPA reported.
Manuel Tapial and Laura Arau - two of the three Spaniards who were on board the ships - lodged a complaint against Israeli Prime Minister Benjamin Netanyahu, six members of his cabinet and a senior military officer at Spain's National Court, DPA reported.
Tapial said his aid association would send two Spanish-flagged ships to Gaza in an attempt to break Israel's blockade.
Israeli naval commandoes shot dead nine activists during the forceful take-over in international waters of the Turkish vessel Mavi Marmara, which along with five other ships had vowed to breach the blockade of Gaza. Israel says its soldiers were attacked by knife and stick-wielding protesters during the operation.
Spain's National Court has pursued human rights cases in Latin America and elsewhere, including failed attempts to try former Chilean dictator Augusto Pinochet and Osama bin Laden.
Its main success was the case of former Argentine navy captain Adolfo Scilingo, whom it sentenced to more than 600 years in prison in 2005 for helping to throw government opponents out of airplanes during the country's 1976-83 military dictatorship.
In April, the court shelved a probe into a 2002 bombing by Israel that killed Hamas leader Salah Shehade and 14 others in the Gaza Strip, after concluding that the case had already been investigated by Israel.