The organisation was notified late on Monday afternoon that access restrictions were mistakenly lifted in a section of the intranet site and a small number of files were opened.
"We are reviewing those files to determine the nature of the access," chief executive Jane Miller said in a statement on Wednesday.
"I trust the integrity of our people and this information has not been accessed outside our organisation."
A third-party IT security provider would complete an audit of the site over the coming days, Ms Miller said.
The matter has been reported to the Office of the Victorian Information Commissioner and the Health Complaints Commissioner as a precaution.
Victorian Ambulance Union secretary Danny Hill said private staff details including medical information, applications and COVID testing information were accessed in the breach.
"These were folders that should have been restricted to senior management and human resources departments and only accessible with authorisation," he told AAP.
"The whole workforce had access to the information."
The latest breach followed a similar incident in May, where the confidential drug and alcohol tests of hundreds of Ambulance Victoria employees and applicants were published on the intranet.
Ambulance Victoria was failing basic standards when it came to protecting staff information, Mr Hill said.
"Repeated failures like this are harmful," he said.
"How can our members feel confident coming forward for mental health support, family violence support, reporting bullying and harassment.
"They can't trust their employer."
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