Trump made his decision after receiving Deputy Attorney General Rod Rosenstein's memo, which cited Comey's handling of the investigation of Hillary Clinton's emails.
But the White House press office found itself deflecting questions about the FBI's investigation into Russia's alleged interference in the 2016 presidential election, which also probes potential connections to the Trump campaign.
"Today's actions have zero to do with Russia," Kellyanne Conway, senior White House advisor, told CNN's Anderson Cooper.
Deputy press secretary Sarah Huckabee Sanders told Fox News that there's no "there there" and that it's "time to move on" from this "false narrative that the media continues to want to drive."
"Frankly, it's kind of getting absurd. There's nothing there. We've heard that time and time again," Sanders said.
Yet after Comey's termination, several Democrats called for the appointment of a special prosecutor, claiming that it is the only way the American people would find the outcome of the probe credible.
In a late Tuesday briefing with reporters, Senate Democratic Leader Chuck Schumer asked why Comey was fired at this particular moment.
"Were these investigations getting too close to home for the president? It is troubling that Attorney General Sessions, who had recused himself from the Russian investigation, played a role in firing the man leading it," Schumer said.
Later Tuesday evening, Trump mocked Schumer in response, citing the New York Democrat's comments to Bloomberg in November that he had lost confidence in Comey.
At the time, Schumer told Bloomberg that he wanted to sit down with Comey to understand why he sent a letter to Congress about the Clinton investigation so close to the election.
Notable Republicans also raised questions about the timing of Comey's dismissal, suggesting a more independent investigation is appropriate.
Trump has repeatedly denied that his campaign colluded with Russia. He has called allegations of ties to Russia a "total hoax" and "an excuse used by the Democrats as justification for losing the election."