Harris assailed Trump's consistent downplaying of the pandemic's threat, insisting she would not take a vaccine if the Republican president endorsed it without the backing of medical professionals.
"Frankly this administration has forfeited their right to re-election based on this," she said.
Less than four weeks before Election Day, Republicans hoped the debate might give the Trump-Pence ticket a final opportunity to help reset a contest that could be slipping away.
His poll numbers sagging, the president, with Pence at his side, is struggling to stabilise the nation in the midst of multiple crises as more than a dozen senior officials across the White House, the Pentagon and inside his campaign have been infected by the virus he claimed would disappear.
There were heated exchanges at times, but overall the debate was a far more respectful affair than the opening presidential debate eight days earlier when Trump was the aggressor, butting in and almost yelling.
Pence interrupted at times, too, but nothing like Trump had.
The prime-time meeting in Salt Lake City elevated two candidates with presidential aspirations of their own who may be asked to step into the presidency even before the end of the next term.
Health questions loom over Trump, 74, who is recovering from the coronavirus, and 77-year-old Joe Biden, who would be the oldest US president ever.
Republicans desperately want to cast the race as a choice between two candidates fighting to move the country in vastly different directions.
Biden and Harris, they say, would pursue a far-left agenda bordering on socialism; the Democrats say Trump's administration will stoke racial and other divides, torpedo health care for people who aren't wealthy and otherwise undercut national strength.
But so long as the coronavirus is ravaging the White House and killing several hundred Americans each day, the election will almost certainly be a referendum on the Trump administration's inability to control the pandemic, which Republicans have sought to downplay or ignore altogether for several months.
Pence's message was undercut by the mere fact that the candidates and moderator were separated by plexiglass shields, seated more than 12 feet apart and facing a crowd of masked audience members who faced expulsion if they removed their face coverings. The candidates on stage revealed test results earlier in the day proving they were not infected.
Before Harris said a word, she made history by becoming the first black woman to stand on a vice-presidential debate stage. The night offered her a prime opportunity to energise would-be voters who have shown only modest excitement about Biden, a lifelong politician with a mixed record on race and criminal justice.
The candidates also clashed on taxes - or specifically, Trump's refusal to release his tax returns four years after repeatedly promising to do so. The New York Times reported last month that the president pays very little personal income tax but owes hundreds of millions of dollars in debt.
"It'd be really good to know who the president owes money to," Harris said.
"The one thing we know about Joe, he puts it all out there. He is honest, he is forthright," she added. "Donald Trump, on the other hand, has been about covering up everything."
Pence defended Trump as a job creator who has paid more than his fair share of taxes and shifted toward Biden: "On Day One, Joe Biden's going to raise your taxes."
While the debate covered a range of topics, the virus was at the forefront.
Trump released a video just three hours before the debate calling his diagnosis "a blessing in disguise" because it shed light on an experimental antibody combination that he credited for his improved condition - though neither he nor his doctors have a way of knowing whether the drug had that effect.
Pence serves as chair of the president's coronavirus task force, which has failed to implement a comprehensive national strategy even as Trump himself recovers from the disease and the national death toll surges past 210,000 with no end in sight.
