The ex-president and former treasurer at the Bambino Gesu in Rome are accused with using the cash to fund a renovation of a senior cardinal's apartment.
Giuseppe Profiti and Massimo Spina have been ordered in stand in a trial which is scheduled to begin on Tuesday next week.
It is alleged they took the sum of money from the paediatric hospital's fundraising foundation to fund improvements at the home of Cardinal Tarcisio Bertone when he retired as the Vatican's secretary of state.
The cardinal has denied knowing about the payment.
Mr Profiti's administration was recently the subject of an Associated Press investigation into quality of care at what is referred to as the "pope's hospital".
He has admitted to the payment but argued it was an investment to allow the foundation use of the apartment for fundraising events.
Bambino Gesu recently caught the attention international news media, after bosses offered to take in terminally ill baby Charlie Gard.