Health Minister Harriet Shing and Energy Minister Lily D'Ambrosio on Thursday announced they would not contest their seats at the November state election and would step down from cabinet.
The exit of the pair, who are prominent allies of Ms Allan's socialist left faction, creates room for new Premier Ben Carroll to reshuffle cabinet in coming days.
Ms Shing said she met with Mr Carroll on Tuesday afternoon, hours after he was sworn in as premier, to notify him of her decision.
The upper house MP was elevated to cabinet in 2022 and promoted to health minister in April after Mary-Anne Thomas flagged her retirement alongside fellow Labor stalwarts Danny Pearson and Gayle Tierney.
The first openly lesbian member of Victorian parliament after her election in 2014, Ms Shing thanked her partner, Â former premiers Daniel Andrews and Ms Allan, as well as Mr Carroll and her caucus colleagues.
"It has always been my view that we are custodians of these roles for the time that we have them, and it is now time for me to pass these opportunities and obligations on to the next person," Shing said.
Representing the seat of Mill Park since 2002, Ms D'Ambrosio was the longest serving energy minister in Australia after her appointment to the role in the first iteration of the Andrews government.
"Back in 2014 not a lot of people thought about energy too much. Of course, the decade that followed changed all that," Ms D'Ambrosio said in a statement.
She informed Mr Carroll she would stand down from the cabinet on Thursday.
"I hope that my life in public service shows all girls what is possible for them no matter their faith, what they look like or the way they speak," Ms D'Ambrosio said in a statement.
"Always back yourselves and each other."
Mr Carroll thanked Ms Shing and Ms D'Ambrosio for their "many years of dedicated service".
He will reshuffle his frontbench following a caucus meeting on Monday and vowed to run a "strong, disciplined and united cabinet".
On Thursday, Mr Carroll hinted he could keep the education portfolio, telling reporters it had always been close to his heart.
"I will have more to say on portfolios," he said.
"I value this portfolio, and this will be a matter for me and discussion with my colleagues."
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