GMB union members at Labour HQ have backed a no-confidence motion in the general secretary, Jennie Formby.
It follows the leaking of an internal report, which included emails and private WhatsApp messages.
The motion calls on Ms Formby to "personally apologise to the current staffers named in the report".
Among its findings, the report claims factions opposed to former leader Jeremy Corbyn hampered efforts to tackle anti-Semitism.
Labour leader Sir Keir Starmer has ordered an inquiry into why the report was commissioned, its contents, and how it came into the public domain.
But in its motion, the GMB, which is the largest union at Labour Party headquarters, accuses Ms Formby of leaking the report, the BBC's Iain Watson reports, and criticises current, as well as former, party managers.
The motion says that by "trawling the emails and instant messenger logs, the general secretary has effectively unilaterally placed all members of staff under investigation" without due process.
And it adds: "Staff can no longer be confident that the general secretary has the safety and welfare of staff as her top priority, and [she] has allowed the mental and physical wellbeing of staff to be put at risk with the creation and leaking of this report."
The motion also refers to a "hostile environment created post-2015", when Jeremy Corbyn was first elected leader, "in which staff who did not appear to support the new leader were marginalised, ignored, harassed and hounded out of the party".
The Unite union, headed by key Jeremy Corbyn ally Len McCluskey, also has a branch at Labour HQ.
It has released a statement saying the report should not have been released unredacted, according to the LabourList website.
But the branch describes as "shocking" the allegations in the report that an anti-Corbyn faction at party HQ worked against a Labour victory at the 2017 general election and warns against the investigation being "kicked into the long grass".
The GMB and Unite branches have previously clashed over their respective responses to the leaked report's contents.