Zapatero's office in Madrid was being searched along with three other premises, the court said in a statement, adding that the former premier had been summoned to testify on June 2.
Zapatero, a key ally of current Socialist Prime Minister Pedro Sanchez, has already denied any wrongdoing before a lower house committee.
The case puts more pressure on Sanchez, who is already dogged by a corruption investigation into alleged kickbacks involving key members of his inner circle, as well as probes involving his wife and his brother.
Sanchez's office did not reply to Reuters' requests for comment.
Zapatero led Spain from 2004 to 2011, winning two absolute parliamentary majorities with landmark policies such as withdrawing Spanish troops from Iraq and legalising same-sex marriage. He is the first Spanish prime minister - current or former - to be formally investigated by the judiciary since the transition to democracy.
In a statement, the Socialist Party said that Zapatero was a pioneer of progressive policies that now underpin Spanish society and that the political right and far right had never forgiven him for this.
The investigation is part of the so-called Plus Ultra case, linked to the 53 million euro ($62 million) state rescue in 2021 of domestic airline Plus Ultra through state holding company SEPI during the COVID-19 pandemic.
The High Court is examining whether the bailout was properly approved. The aid has drawn political fire as critics said it was not clear that Plus Ultra was a strategically significant asset and that it had weak finances and links to Venezuelan shareholders.
The conservative opposition People's Party (PP) has repeatedly criticised Zapatero's business ties in Venezuela in the years following his departure from government.
The PP on Tuesday described Zapatero as Sanchez's "muse" and said the two allies were linked by corruption allegations.
"Both have used their families to enrich themselves and both have denigrated the institution they represent or have represented," the right-wing party said in a statement.
($1 = 0.8597 euros)
(Reporting by David Latona; Editing by Charlie Devereux and Hugh Lawson)

National Police Agents gather confiscated files and material into a vehicle of the Spanish Central Operative Unit during a police raid at the office of Spanish former Prime Minister Jose Luis Rodriguez Zapatero in Madrid, Spain, 19 May 2026. A judge from the Audiencia Nacional Court has ordered a formal investigation on former Prime Minister Jose Luis Rodriguez Zapatero in the Plus Ultra case that investigates a 53 million euros state bailout back in March 2021. EPA/Rodrigo Jimenez