The agency provides Palestinian refugees and their descendants across the Middle East with services including schools and medical care, but Prime Minister Benjamin Netanyahu has long accused it of hostility toward Israel and called for its closure.
Some five million Palestinians are eligible for its services.
On Tuesday, the United States held back UD 65 million that had been destined for the agency, two weeks after President Donald Trump threatened future payments.
"The US has announced it will contribute $60 million to the programme budget. There is for the moment no other indication of possible funding," UNRWA spokesman Chris Gunness told AFP.
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"This dramatically reduced contribution results in the most severe funding crisis in the history of the agency."
He added that "stability in the Middle East is what we are talking about and the potential impact of UNRWA being destabilised is very likely to be widespread, profound, unpredictable and catastrophic."
Senior Palestinian officials reacted with outrage to what they see as another move against them by Trump's White House following his declaration of Jerusalem as Israel's capital.
The Palestinian envoy to Washington, Husam Zomlot, said: "Palestinian refugees and children's access to basic humanitarian services, such as food, health care and education, is not a bargaining chip but a US and international obligation."
Palestinian officials also accused Trump of chipping away at issues long considered up for negotiation as part of a comprehensive resolution to the Israeli-Palestinian conflict, including the status of Jerusalem and the plight of refugees.
Around 500 people protested in the Gaza Strip on Wednesday against the freeze.
France meanwhile said it "regrets" the US move and was in contact with Washington about it.
The funding freeze comes with relations between the Palestinians and Washington already on the brink.
On Sunday, Palestinian president Mahmud Abbas denounced Trump's peace efforts as the "slap of the century," while Palestinian leaders have threatened to suspend their recognition of Israel.
US State Department officials insisted the decision to freeze the funding was taken not to pressure Palestinian leaders but to encourage other countries to help pay for and reform UNRWA.