In previously unheard testimony, Bill Taylor, the acting US ambassador to Ukraine, said a member of his staff was told Mr Trump was preoccupied with pushing for a probe into Mr Biden.
He was speaking at the first public hearings in the impeachment inquiry.
Mr Trump told reporters he did not recall making such comments.
Mr Trump is accused of withholding US military aid to Ukraine in order to pressure the country’s new president to publicly announce a corruption inquiry into Mr Biden, among the favourites to take him on in the 2020 presidential race.
Mr. Trump denies any wrongdoing and has called the inquiry a “witchhunt”.
What did Trump allegedly ask about?
During a detailed opening statement, Mr Taylor said a member of his staff had overheard a telephone call in which the president inquired about “the investigations” into Mr Biden.
The call was with Gordon Sondland, the US ambassador to the European Union, who reportedly told the president over the phone that “the Ukrainians were ready to move forward”.
After the call, the staff member “asked ambassador Sondland what President Trump thought about Ukraine”, Mr Taylor said.
Mr Taylor said: “Ambassador Sondland responded that President Trump cares more about the investigations of Biden.”
When asked about Mr Sondland earlier this month, the president had said: “I hardly know the gentleman.”
Responding to queries from reporters after the hearing, Mr Trump said: “I know nothing about that, first time I’ve heard it.”
He said he recalled Mr Sondland’s testimony, in which the diplomat said he spoke to the president “for a brief moment” and Mr Trump had “said no quid pro quo under any circumstances”.
Mr Sondland he did not recall the phone call Mr Taylor described, “not even a little bit”, and “in any event it’s more second hand information”, he said.
The impeachment inquiry has been going on for more than a month – but all previous hearings were private, with reports based on leaks and sources speaking to the media.
Wednesday’s public hearings were the first time the public heard from witnesses directly and a chance for Democrats and Republicans to win over voters.
Why the new information matters
This has the potential to be a major twist. Although there have been reports of Mr Sondland’s direct line to the president, there has yet to be evidence tying Mr Trump directly to the alleged quid pro quo.
The phone call Mr Taylor described could change all that.
In the middle of Wednesday’s hearing, the House Intelligence Committee announced a new witness scheduled to give a closed-door deposition on Friday, an aide named David Holmes – reportedly the aide Mr Taylor mentioned.
Next week, Mr Sondland himself is scheduled to testify during public hearings.
If either of these two men support Mr Taylor’s account, it could undercut the president’s defenders who have suggested that Mr Trump was not closely involved in the activities of the “unofficial” channel of Ukraine policy, as Mr Taylor called it, which was pressuring Ukraine to open up investigations into the Bidens.
Democrats have reason to be pleased, while the president’s team has a new set of headaches.
What else happened at the hearing?
Wednesday’s hearing began with testimony from George Kent, a top US diplomat charged with overseeing European affairs.
He told the committee that President Trump’s private lawyer, Rudy Giuliani, ran a “campaign to smear” the US ambassador to Ukraine, Marie Yovanovitch, before she was recalled from her post.
Meanwhile, much of Mr Taylor’s other testimony repeated evidence he gave to private Congressional impeachment hearings, but he added new detail to the accounts in the public domain.
The career diplomat, who has served under Republican and Democratic presidents, reiterated his understanding that the Trump administration threatened to withhold military aid from Ukraine unless the Ukrainian president agreed to publicly announce an investigation into Mr Biden.
He testified that he told Mr Sondland and Kurt Volker, previously the US special envoy to Ukraine, that it would be “crazy” to withhold security assistance for the sake of domestic politics.