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The bill included a $158,000 payment to the former staffer to settle the defamation claim - and more than $175,000 was spent on legal fees.
Mallard issued a public apology to the staffer this week, which was released the afternoon of the Royal Commission of Inquiry into the Christchurch terror attacks.
The Speaker said that some of his comments gave the "impression that allegations made against that individual in the context of the Francis Review amounted to rape".
"Trevor Mallard accepts that his understanding of the definition of rape at that time was incorrect and that the alleged conduct did not amount to rape (as that term is defined in the Crimes Act 1961) and that it was incorrect of him to suggest otherwise," the statement said.
"Trevor Mallard apologises for the distress and humiliation his statements caused to the individual and his family."
The cost of settling the defamation case settlement were released to National in answers to written parliamentary questions.
National leader Judith Collins said the party had now lost confidence in Mallard.
"This is unacceptable behaviour from the Speaker of the House. This sheer size of this pay-out illustrates how serious the matter is," Collins said.
"It is the Speaker's job to set the standard of behaviour for everyone at Parliament but he has been reckless with his words, resulting in taxpayers footing a bill of more than $330,000 to clean up this mess.
"There has been no formal apology to Parliament for this, despite the National Party encouraging the Speaker to do so on the final sitting day this year.
"Because Mr Mallard has not lived up to the high standards of behaviour that he has set for Parliament, we believe he is no longer fit to hold the role of Speaker.
"The people who work at Parliament, and the taxpayers of New Zealand, deserve better."
How the saga unfolded
In June last year, Mallard said that he believed a rapist was working in the parliamentary precinct a day after the Francis review of the parliamentary workplace was released.
The report found systemic issues of bullying and harassment, poor conduct too often tolerated and normalised, and a perception of low accountability. Mallard himself commissioned the report after a series of cases of bad behaviour.
Mallard had said that he believed a man was responsible for three serious sexual assaults mentioned in the review, and that he believed the man was still working at Parliament.
His comments - called shocking by some parliamentary workers - sparked a series of turbulent events that led to a historical assault complaint and a parliamentary staffer being stood down.
Mallard then said that a threat to safety had been removed from the premises.
The staffer then lashed out at Mallard, saying he felt bullied out of the workplace and was the victim of Mallard's "slanderous" comments.
After Mallard made the comments, the man - who had been stood down from Parliament - told Newstalk ZB he felt bullied out of the building.
He said at the time he wanted an apology for what he described as the Speaker's "slanderous" comments.
The man claimed the three allegations were related to hugging a colleague, complimenting another colleague on her hair, and kissing another on her cheek as he said goodbye to her after she visited him and his wife for tea.
However, the first complainant alleged he hugged her from behind, pushing his groin up against her, and that he was staring at the breasts of the woman who's hair he complimented.
He believes the third complainant was put up to lodging the complaint by someone else.
The man was investigated by Parliamentary Services, which found the claims were unsubstantiated.