Department of Health and Human Services secretary Kym Peake is the third key witness to quit after appearing at Victoria's hotel quarantine inquiry.
It follows former Health Minister Jenny Mikakos and the state government's most senior public servant, Department of Premier and Cabinet secretary Chris Eccles, resigning from their roles.
Ms Mikakos quit in late September after strongly disagreeing with elements of Premier Daniel Andrews' evidence to the inquiry, in which he said she was accountable for the program.
A little over a fortnight later, Mr Eccles handed in his resignation when a review of his phone records revealed he'd spoken to then-police boss Graham Ashton as the fateful decision was made to use private security guards.
Ms Peake's decision to quit comes ahead of the inquiry, headed by former judge Jennifer Coate, handing down its final report by December 21.
Before its final sitting on October 20, the inquiry heard Ms Peake received an email on April 9 warning of a "risk to the health and safety of detainees" due to governance issues.
It was written by Public Health Commander Finn Romanes and backed by Chief Health Officer Brett Sutton and his deputy Annaliese van Diemen.
Ms Peake and her deputy Melissa Skilbeck also denied a request from Premier Daniel Andrews to house passengers from the Greg Mortimer cruise ship at a hotel near the airport.
Eighty of the passengers were COVID-positive.
The passengers were instead sent to the Rydges on Swanston. Along with the Stamford Plaza, the inquiry was told about 99 per cent of the state's second wave could be traced back to those two hotels.
In addition, Ms Peake failed to brief Ms Mikakos on two Safer Care Victoria reports that identified problems with the botched program.
"I'm actually very disappointed that they were not raised with me," Ms Mikakos told the inquiry.
Both she and Ms Mikakos described the scheme as a "multi-agency operation with shared accountability".
"I know it would be more straightforward if there was an ability today to say there's a single accountability ... but I do think the whole weight of the evolution of public service delivery is that people are not carved up into portfolios," Ms Peake told the inquiry on September 22.
Ms Peake said the jobs department managed hotels, security, food, cleaning and the guest helpline, while the health department was responsible for health and wellbeing.
Asked if she accepted deficiencies in the hotel quarantine program stemmed from failures in her department, Ms Peake said: "No I don't."
"It is a matter of profound regret to me as the secretary of the Department of Health and Human Services that we experienced a second wave in Victoria and all of the consequences that came with that."
The state's second outbreak resulted in more than 18,000 new infections and 750 deaths.
Professor Euan Wallace has been hired as Ms Peake's replacement and will start in the new role from next week.
The professor of obstetrics and gynecology at Monash University has been on secondment from his role as chief executive at Safer Care Victoria to serve as deputy secretary at the DHHS.
In a statement, the Victorian government said Ms Peake resigned to pursue "other opportunities".