St Kevin's College deputy head Janet Canny has been stood down following allegations she responded inappropriately to a teacher's complaints about child-grooming allegations.
Edmund Rice Education Australia (EREA) confirmed on Thursday that Mrs Canny had been removed from her position.
The decision follows the resignation of St Kevin's headmaster Stephen Russell on Wednesday and the standing down of dean of sport Luke Travers over the school's handling of a 2014 child-grooming case.
The Melbourne Catholic boys' school is set to undergo more scrutiny after teacher Maree Keel alleged she was mistreated and has launched legal action under the Fair Work Act.
"Ms Keel alleges she was mistreated after raising concerns about the behaviour of a St Kevin's staff member," law firm Maurice Blackburn said.
The ABC reported on Wednesday that St Kevin's allegedly responded inappropriately to Ms Keel's complaint about inappropriate sexual conduct by a fellow teacher last year.
Mrs Canny told Ms Keen she was being "disloyal to St Kevin's College", the ABC said.
Mr Russell had been under pressure after it was revealed he provided a character reference to a court for former volunteer athletics coach Peter Kehoe who was convicted of grooming a student under 16.
Premier Daniel Andrews said St Kevin's parents had every right to be unhappy with Mr Russell's actions and welcomed his resignation.
"If he'd been a principal at a government school, I wouldn't have had confidence in him. So I think that parents at St Kevin's have got every reason to be not pleased by his conduct, (but) perhaps pleased by his most recent decision."
Mr Travers, who also gave Kehoe a reference, was stood down, pending an investigation.
St Patrick's College Ballarat principal John Crowley will take over as headmaster on Friday until the end of the school year.
In a second statement on Thursday, EREA also announced that James Daly would be the acting deputy head at St Kevin's.
He is already director of the school's Glendalough junior campus.
Mr Daly will be the school's interim senior child safety officer.
The college board will be reconstituted, with Paul O'Malley as its new chairman.