Paya, a joint Cuban-Spanish national, and another Cuban dissident, Harold Cepero, died on July 22, 2012 in southeastern Cuba after their car went off the road and crashed into a tree.
Paya's wife Ofelia Acevedo and his daughter Rosa Maria Paya presented a suit at Spain's high court against two state security officials named as Lieutenant Colonel Aguilas and Colonel Llanes, said a statement from Paya's rights group, the Christian Liberation Movement.
The lawsuit also targets anyone else "that the court might find, in the course of its investigation, to have taken part in the crimes against humanity that led to the death of Oswaldo," the statement said.
"This suit is necessary and indispensable," it added.
"The current Cuban regime cannot continue persecuting, mistreating, lying, assassinating and intimidating society with impunity."
The driver of the car, Angel Carromero, a leader of the youth wing of Spain's ruling Popular Party, was convicted in Cuba of vehicular homicide. He returned to Spain to serve a four-year sentence and was granted conditional release.
Carromero alleged in a newspaper interview published this month that the crash was not an accident and that Paya was "assassinated" by the Cuban secret service.
Oswaldo Paya was the lead organiser of the Varela Project, a drive for a referendum on guaranteeing rights such as freedom of speech and assembly in Cuba.
His death at 60 deprived the Cuban opposition movement of its main leader.