The televised encounter in Nashville, Tennessee, represented one of Trump's last remaining opportunities to reshape a race that national opinion polls show he has been losing for months, though the contest is much tighter in some battleground states likely to decide the election.
"Anyone who's responsible for that many deaths should not remain president of the United States of America," Biden said.
Trump, who has put his stewardship of the economy at the center of his campaign, defended his approach to the outbreak and said the country could not afford to close businesses again despite fresh surges.
"We're learning to live with it," said Trump, who has played down the virus for months. "We have no choice."
"Learning to live with it" Biden retorted. "Come on. We're dying with it."
Trump asserted that the virus was "going away," several U.S. states reported record single-day increases in COVID-19 infections on Thursday, evidence the pandemic is accelerating anew.
Trump, whose instinct remains to run as an outsider, portrayed Biden as a career politician whose nearly 50-year record was insubstantial. But he did not lay out a clear agenda for a second term, while Biden returned again and again to Trump's four years as president, pointing to the economic damage the virus has done to people's lives.
After an opening segment on the pandemic, Thursday's clash pivoted to rapid-fire exchanges over whether either candidate had improper foreign entanglements.
Trump repeated his accusations that Biden and his son Hunter engaged in unethical practices in China and Ukraine. No evidence has been verified to support the allegations, and Biden called them false and discredited.
Trump's effort to uncover dirt on Hunter Biden's Ukraine business ties led to the president's impeachment. The president and his children have been accused of conflicts of interest of their own since he entered the White House in 2017, most involving the family's international real estate and hotel businesses.
Biden defended his family and said unequivocally that he had never made "a single penny" from a foreign country, before pivoting to accuse Trump of trying to distract Americans.
"There's a reason why he's bringing up all this malarkey," Biden said, looking directly into the camera. "It's not about his family and my family. It's about your family, and your family's hurting badly."
He accused Trump of avoiding paying taxes, citing a New York Times investigation that reported Trump's tax returns show he paid almost no federal income tax over more than 20 years.
"Release your tax returns or stop talking about corruption," Biden said.
Trump, who has broken with decades of presidential precedents in refusing to release his tax returns, said he had paid "millions." He again said he would release his returns only once a longstanding audit was completed.
The candidates argued over foreign policy, immigration and - after months of anti-racism protests - race relations, with Biden saying Trump was "one of the most racist presidents" in history.
"He pours fuel on every single racist fire," Biden said. "This guy has a dog whistle as big as a foghorn."
Trump responded by criticizing Biden's authorship of a 1994 crime bill that increased incarceration of minority defendants while asserting that he had done more for Black Americans than any president with the "possible" exception of Abraham Lincoln during the U.S. Civil War in the 1860s.
OIL IN SPOTLIGHT
During a segment on climate change, Biden said his environmental plan would "transition from the oil industry" in favor of renewable energy sources, prompting Trump to go on the attack.
"He is going to destroy the oil industry," Trump said. "Will you remember that, Texas Will you remember that, Pennsylvania"
Biden said he simply wanted to eliminate federal subsidies for oil companies, a point he reiterated to reporters following the debate. "We're not getting rid of fossil fuels for a long time," he said.
During the debate, Biden criticized Trump's effort to persuade the U.S. Supreme Court to invalidate the 2010 Affordable Care Act, the sweeping healthcare reform passed when Biden was vice president in President Barack Obama's administration.
"People deserve to have affordable healthcare, period," Biden said, noting that the law prevented insurance companies from denying coverage to people with pre-existing conditions.
Trump said he wanted to replace the ACA with something "much better" that would offer the same protections, even though the administration has yet to propose a comprehensive healthcare plan despite a promise to do so for years.
Relatively few voters have yet to make up their minds, and Trump's window to influence the outcome may be closing. A record 47 million Americans already have cast ballots, eclipsing total early voting from the 2016 election.
The contentious first debate, when the two men traded insults, was watched by at least 73 million viewers. Trump passed up another planned debate last week after it was switched to a virtual format following his COVID-19 diagnosis.
(Reporting by Trevor Hunnicutt and Jeff Mason; Additional reporting by Doina Chiacu, James Oliphant and Jason Lange; Writing by Joseph Ax; Editing by Soyoung Kim and Howard Goller)