Public school staff across Victoria will walk off the job on Thursday following a vote by the Australian Education Union's state branch on Tuesday.
Teachers will down tools and also refuse to work unpaid overtime unless an eleventh-hour agreement can be struck with the state government.
It comes after the powerful teacher's union in June knocked back the Labor government's offer of a 28 per cent pay rise over four years.
Branch president Justin Mullaly said teachers, faced with untenable workloads and uncompetitive pay, had been left with no choice.
"In this underfunded system, teachers, principals, and education support staff are working an average of 12 hours unpaid overtime every week," he said.
"The government must stop relying on the goodwill of school employees as a core part of their funding model for schools."
The government urged the union to return to negotiations to prevent disruptions to families across the state.
"This deal, which was endorsed by the AEU leadership, would have made Victorian teachers the best paid in the country together with the best conditions," a government spokesperson said.
"We will always back our hardworking teachers, school leaders and education support staff."
Teachers previously walked off the job in March, when staff took to the streets of Melbourne in the tens of thousands dressed in AEU red.
As many as 35,000 people marched to the front steps of state parliament during the strikes, the first of its kind in more than 13 years.
Other forms of industrial action remain in force, including a ban on state Labor MPs visiting public schools and responding to education department emails.
Mr Mullaly said working conditions had left many educators weighing up leaving the profession they loved just to make ends meet.
"Just three in 10 employees expect to remain working public schools until retirement and report that excessive workloads are one of the reasons why they will leave," he said.
"There is already a chronic shortage of teachers, which the state government has not adequately addressed."
The state branch represents more than 60,000 public school teachers, principals and support staff.
In June, Education Minister Ben Carroll accused the union's leaders of being out of touch with members after they agreed to the government's in-principle deal in June but failed to get it over the line with members.
He warned the offer might not be as generous if a Liberal or One Nation government was elected in five months at the state poll.
Opposition education spokesman Brad Rowswell said the strike showed Mr Carroll had failed again and he must do his job and get a deal done.
"Victorian families shouldn't be paying the price for Labor's failure to manage the state's finances or negotiate with the workforce that educates our children," he said in a statement on Tuesday.
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