In response, GB News chief executive Angelos Frangopoulos said the fine was a “direct attack on free speech and journalism in the United Kingdom”.
“We believe these sanctions are unnecessary, unfair and unlawful,” he added.
The hour-long show, which aired on 12 February, saw members of the public put questions to the then-prime minister.
However, Ofcom received 547 complaints about the programme.
The regulator found earlier this year that while featuring Mr Sunak was fine in principle, “due weight” should have been given to an “appropriately wide range of significant views” other than the Conservatives.
It said the then-prime minister “had a mostly uncontested platform to promote the policies and performance of his Government in a period preceding a UK General Election,” which it recorded as a breach of impartiality rules.
On Thursday, Ofcom said “given the seriousness and repeated nature of this breach,” it has imposed a £100,000 financial penalty on GB News.
Mr Frangopoulos said in response that the programme “was an important piece of public interest programming” and that “appropriate steps” were taken to ensure due impartiality.
He added: “It was designed to allow members of the public to put their own questions directly to leading politicians.
“GB News chooses to be regulated and we understand our obligations under the Code.
“But, equally, Ofcom is obliged by law to uphold freedom of expression and apply its rules fairly and lawfully.”