A meeting of around 40 Bolton Unison representatives overwhelmingly supported a no confidence resolution on Cllr Cliff Morris and town hall's wider leadership at a meeting on October 5.
The union, which represents thousands of Bolton Council workers, will now hold a further vote among its membership.
This is the latest blow to Cllr Morris' leadership, following a vote of no confidence that was passed by the Bolton West Constituency Labour Party.
Bernadette Gallagher, Bolton Unison branch secretary, said: “Our members have borne the brunt of seven years of austerity and cuts that have been implemented by Bolton Council.
"There is widespread disaffection and anger within Bolton towards the actions of Cliff Morris, including the recent use of emergency powers to force through a £300,000 grant to law firm Asons which severely damaged the council’s reputation.
"This dissatisfaction and anger is felt by council workers in workplaces right across the town."
She added: "It is not surprising that our union representatives feel so strongly that they now have no confidence in the leader of the council, who is ultimately responsible for council decisions.
"The buck stops with him and it is not surprising that there is a growing view that Cllr Morris should now step aside for the good of the council, the workforce and the town. However this is not just an issue about one individual, it is an issue about the wider leadership within the council."
Cllr Morris declined to comment.
Last month, two senior Bolton Labour officials were ousted from their posts amid a row over a councillor who was at the centre of a town hall tax scandal.