The former special forces soldier said Pauline Hanson and One Nation MP Barnaby Joyce were "lying cowards" and called the party leader "glass-jawed" after a cartoon video was posted to her X account which took aim at Mr Hastie.
Politicians of all stripes, including Opposition Leader Angus Taylor, have demanded the One National leader remove the video, which remains pinned to the top of her profile.
"Andrew Hastie has served this country with distinction as an officer in the SAS, she should take it down," Mr Taylor told reporters on Thursday.
Mr Hastie had accused One Nation of politicising the court proceedings of accused war criminal Ben Roberts-Smith and "trashing" his own service record.
The WA-based MP was subpoenaed to testify against Roberts-Smith's in a defamation trial in 2022, which generated tension between himself and Senator Hanson.
The spat comes as the coalition seeks to nullify a surge of support for Senator Hanson's populist party, which has split much of the right-wing vote especially in regional and rural Australia.
The WA state seat of Secret Harbour, which sits squarely within Mr Hastie's federal seat of Canning, was the latest to flip to One Nation at a by-election in August, as the anti-immigration party eyes off coalition-held electorates.
Senator Hanson on Wednesday denied calling the Liberal MP a traitor, saying it was only a satirical cartoon and he was "making a mountain out of a molehill".
But the Liberal MP, seen by some political commentators as the party's potential future leader, said the video had cast a "massive slur" on veterans across the country and he would continue to call out the One Nation leader.
"It shows her to be weak and glass-jawed. I gave the opportunity to either own her words publicly, or to take it down, she chose neither," he told ABC Radio.
"Rather than stepping up as a leader, she claims she could be prime minister, she actually retreated back into cartoons and memes.
"So I think we've seen what Pauline Hanson ultimately is about. She's not fit to govern this country."
But a chorus of political adversaries joined Mr Hastie in condemnation the attacks from One Nation, including Tasmanian senator and fellow army veteran Jacqui Lambie.
"She's disgusting ... she's the lowest bit of life you can possibly come from with that woman," Senator Lambie said of the One Nation leader, also calling for the cartoon of Hastie to be taken down.
"You watch this, the Australian people, here we go, here comes coward Hanson."
While he said he disagreed with most of what Mr Hastie says, Greens senator David Shoebridge also came to the Liberal's defence, describing the attack as disgraceful.
"You might agree or disagree, but (Hastie) goes into court and tells the truth as they understand it under oath, bloody hell, that's actually what we want our politicians to do," he told News24.
Mr Hastie said he was not seeking legal action against Senator Hanson, citing concerns about One Nation leader's support from billionaire mining magnate Gina Rinehart.
Rinehart, who is Australia's richest person, is a fierce supporter of Roberts-Smith and has named a planned housing complex for veterans in his honour.
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