"Failure to act would hand a victory to (Russian President Vladimir Putin) and have unthinkable consequences not only for Ukraine, but also for our collective security," Yellen said.
Speaking at the start of a Washington meeting with EU executive vice presidents Margrethe Vestager and Valdis Dombrovskis, she added that Congress must act quickly to reach a deal.
"We cannot allow Ukraine to run out of money while it continues its valiant struggle to defend its democracy," she said.
Yellen's remarks come as US lawmakers struggle to reach agreement on budget funding, including support to Ukraine and Israel, and for substantial immigration reform.
On Wednesday, Democratic Senate leader Chuck Schumer said on the Senate floor: "The survival of Ukraine is on the line."
As fears surround the issue of further military aid from the United States, Brussels has also been demanding clear figures from EU states on what they are giving Kyiv this year.
On Wednesday, the European Union said it would supply only half of the million artillery rounds it had pledged for Ukraine by March.
Vestager and Dombrovskis, who were in Washington for a meeting of the US-EU Trade and Technology Council, stressed the need to keep up the transatlantic partnership.
"We must stay the course, we must keep pressure on Russia," Dombrovskis said, adding that a part of this means imposing and enforcing sanctions on Moscow.
Vestager added at the start of their meeting with Yellen that "we're in it for the long haul," and flagged the importance of efforts to provide 50 billion euros ($54 billion) in budget support for Kyiv over the coming years.
But for now, talks over EU military support for Ukraine are rolling over into a summit of European leaders this week, with Hungary blocking the package.
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