The bank, officially known as the Institute for Religious Works (IOR), said it had pressed charges against the trio some months ago and "the accounts held by the concerned individuals at the IOR have recently been seized".
Italian media reports named the accused as former bank president Angelo Caloia, ex-director general Lelio Scaletti, and lawyer Gabriele Liuzzo.
While the IOR would not provide details on the case "given the ongoing judicial enquiry", Vatican spokesman Federico Lombardi told Italian media they were suspected of embezzling money.
In a statement the bank said the former managers and lawyer were under investigation based on "circumstances recorded between 2001 and 2008 that have emerged in the internal review process initiated in early 2013."
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The IOR has been trying to rid itself of a reputation for shady dealings, following a series of money-laundering scandals in the past.
It was the main shareholder of the Banco Ambrosiano, which collapsed in 1982 amid accusations of ties to the Mafia, while its chairman Roberto Calvi -- dubbed "God's Banker" -- was found hanging from Blackfriars Bridge in London in a suspected murder by mobsters.
"We are very pleased that the Vatican authorities are taking decisive action," Jean-Baptiste de Franssu, head of the IOR board, was quoted as saying.