The European Public Prosecutor's Office (EPPO) said the investigation focused on "suspected fraud related to EU-funded training for junior diplomats".
It involved searches at the EU's diplomatic service in Brussels, at the College of Europe - an elite university in Bruges that educates many EU officials - and at the houses of suspects.
Mogherini and senior diplomat Stefano Sannino, both Italians, are well known in Brussels diplomatic circles and news of their detentions sent shockwaves through the EU community in the city.
Mogherini, a former Italian foreign minister, was the EU's high representative for foreign and security policy and head of its diplomatic service from 2014 to 2019.
She became rector of the College of Europe in 2020.
Sannino previously served as the most senior civil servant in the diplomatic service between 2021 and 2024 and is now director general of the European Commission's department for the Middle East, North Africa and the Gulf.
The EPPO said in a statement that three people had been detained but withheld identities, citing the ongoing investigation.
Neither Mogherini nor Sannino could be reached for comment.
The prosecutor's office said its investigation centred on the establishment of the European Union Diplomatic Academy - a nine-month training program for junior diplomats, which was awarded to the College of Europe in 2021-2022 by the EU diplomatic service following a tender procedure.
It said there were "strong suspicions" that confidential information was shared during the process with one of the candidates participating in the tender.
"Prior to the searches, the EPPO requested the lifting of the immunity of several suspects, which was granted," the statement said.
"All persons are presumed innocent until proven guilty by the competent Belgian courts of law."
A spokeswoman for the EU diplomatic service confirmed police visited its offices on Tuesday as part of an investigation into activities that took place before the current foreign policy chief Kaja Kallas took office.
The diplomatic service is "fully co-operating with the authorities," spokeswoman Anitta Hipper said.
"Since it's an ongoing investigation, we will not be able to say more."
The College of Europe said in a statement it would "fully co-operate with the authorities in the interest of transparency and respect for the investigative process".
"The College remains committed to the highest standards of integrity, fairness, and compliance - both in academic and administrative matters," it said.
with AP and DPA
