Police spokesman Brad Battin, first-term MP Jess Wilson and Mornington MP Chris Crewther have announced their candidacy if a motion to challenge Mr Pesutto is successful.
Before that takes place, the party must determine whether absent MPs can vote, which will be followed by a second vote on whether to return banished colleague Moira Deeming to the fold.
After this, a spill motion is expected to be raised.
Former party leader Michael O'Brien told reporters he would rather be at the cricket than in the party room but underscored the need for unity.
"Unless we can win and hold government, then we can't enhance the freedom, prosperity and security of Victorians or Australians," Mr O'Brien said as he entered Parliament House..
"So we just need to knuckle down, get behind whatever decision is made today and win the next election for Victoria."
Jess Wilson is prepared to contest the leadership "to provide Victorians with a real choice". (Joel Carrett/AAP PHOTOS)
Opposition financial spokeswoman Jess Wilson announced her candidacy after a deal to become leadership frontrunner Brad Battin's deputy came off the table.
"The best way forward to defeat Jacinta Allan and Labor was with a unified leadership ticket," Ms Wilson said.
"Unfortunately, it has been made clear to me that a unity ticket is no longer on the table.
"Given that and after consulting my colleagues, I've decided to stand to offer them a choice."
Mr Battin arrived for the vote flanked by supporters including Sam Groth, while Ms Wilson and Mr Pesutto entered parliament alone.
Mr Pesutto entered the opposition party room with deputy David Southwick saying his thoughts were with Victorians experiencing bushfires and the first responders.
"We'll deal with these matters in here but the most important thing for us in Victoria is to look out for each other," he said.
Brad Battin is the frontrunner to depose John Pesutto as opposition leader. (Joel Carrett/AAP PHOTOS)
Mr Crewther's eleventh-hour tilt at the top job came via an email to party members overnight, urging collegues for a "fresh start".
"I hope that after today, whoever is chosen as a leader, we all unite behind that person, we all work together, because that's what the Victorians want, they don't want a divided team," he said entering the opposition party room.
Mr Pesutto's job had been on shaky ground ever since he lost a defamation case brought against him by former colleague Moira Deeming.
A Federal Court judge found the Liberal leader defamed Mrs Deeming by implying she was associated with Nazis who gatecrashed a controversial Melbourne rally she was at and ordered he pay her $315,000 and costs.
Then came a shock when former tennis player-turned-politician Mr Groth quit his shadow cabinet post, citing his leader's refusal to stand down after the court loss.
A party vote, brought on less than a week later to decide whether Mrs Deeming would be allowed back into the party, split the team down the centre with Mr Pesutto casting the tie-breaking vote to bar her from returning.
Mr Battin (2nd right) arrived for the meeting with a supporting group of MPs. (Joel Carrett/AAP PHOTOS)
The Hawthorn MP deemed the matter "resolved" only to propose a January 15 gathering to return the upper house MP to the fold a day later.
But that last bid to quell tensions fell flat, with senior MPs Mr Groth, Richard Riordan, James Newbury, Mr Battin and Bridget Vallence signing a petition to meet on Friday to bring the issue to a head.
The battle to helm Victoria's opposition comes less than two years before a pivotal state election at which the Labor government - increasingly on the nose with voters - will seek a rare fourth term.
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