Sally McManus has been secretary of the Australian Council of Trade Unions (ACTU) since 2017, and Michele O'Neil its president since 2018.
The pair announced on Monday via a statement they would leave their posts, making way for younger leaders to "take (the) movement forward".
New leadership is yet to be decided.
''The ACTU's union affiliates will decide on the next leadership team, initially with the ACTU Executive filling the casual vacancies and then through an election at next year's Congress, which will coincide with the ACTU's 100th anniversary," the statement said.
Ms McManus will finish in the role at the end of August, while Ms O'Neil will depart before the end of the year to ''assist with the leadership transition''.
While the pair have been praised for their work in securing deals for workers, the ACTU has been plagued by difficulty after alleged infiltration of criminals in the Construction, Forestry and Maritime Employees Union (CMFEU).
Ms McManus in 2024 denied she knew any of the behaviour in the Victorian branch, and also oversaw the ACTU's suspension of CFMEU divisions while it sorted itself out.
It was "essential" the sector emerged from that turbulent period under new leadership, Ms O'Neil told ABC RN on Tuesday.
"Workers who work in the construction industry around this country, by and large, are hardworking, good people who work in tough and dangerous jobs, and they deserve a great union.
"They deserve a union that is completely free of any violence or corruption or organised crime," she said.
Prime Minister Anthony Albanese thanked the pair for their "tireless advocacy" for workers, and their support to him personally.
"It is fitting that Sally and Michele announce they're stepping down from their respective positions at a time when there have been significant wage increases in critical and long-undervalued sectors, the gender pay gap is at a record low and union membership is growing for the first time in decades" he said in a statement.
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