Ms Gabbard confronted the Republican party member about his admission to “embellishing” his resume and fabricating details about his college, finances and employment at prominent institutions such as Goldman Sachs and Citigroup.
“If I were one of those in New York’s 3rd District right now, now that the election is over, and I’m finding out all of these lies that you’ve told, not just one little lie or one little embellishment — these are blatant lies,” Ms Gabbard said while filling in for Fox News host Tucker Carlson on Tuesday.
“My question is — do you have no shame?”
“And the people who are now, you’re asking to trust you to go and be their voice for them, their families and their kids in Washington?”
Mr Santos responded by saying that president Joe Biden had been lying to Americans for “40 years” and the Democrats “resoundingly support him. Do they have no shame?”
Ms Gabbard interjected, adding that her question was not about the Democratic party.
“You don’t seem to be taking this seriously. You have apologised, said you have made mistakes but a lie is not an embellishment on a resume. These are blatant lies,” she said.
“It draws into question how your constituents and the American people can believe anything you may say on the floor of the House.”
Mr Santos claimed some of the allegations about his employment history were “debatable” and that he was willing to explain himself.
He previously drew fire for representing himself as an “American Jew” and claiming his grandparents fled Europe during the Second World War.
When pressed about his heritage, Mr Santos said: “My heritage is Jewish. I’ve always identified as Jewish. I was raised as a practicing Catholic.”
“Not being raised a practicing Jew, I’ve always joked with friends and circles, even within the campaign, I’d say, guys, I’m ‘Jew-ish.’ Remember, I was raised Catholic.”
“So, look, I understand everybody wants to nitpick at me. I’m gonna reassure this once and for all. I’m not a facade. I’m not a persona. I have an extensive career that I worked really hard to achieve.
“And I’m going to deliver from my experience because I remain committed in delivering results for the American people,” Mr Santos added.
Earlier in the day, the Republican Jewish Coalition, a powerful GOP group, released a statement condemning the misrepresentations of his supposed Jewish faith and barred him from future events.
Meanwhile, Democrats in Congress have demanded Mr Santos face a House Ethics Committee investigation, something that could occur once he takes office in January.