The House voted to deny funding for direct help to the rebels although a proposal to stop US participation in Nato-led air strikes was defeated.
Correspondents say there is strong Congressional opposition to US involvement in the conflict.
Opponents say President Obama should have sought Congress's approval first.
The House voted 225-201 to stop any money from the defence spending bill being spent on equipment or training for rebels trying to oust Libyan leader Col Muammar Gaddafi.
The measure needs Senate approval and to be signed by President Obama before becoming law.
Republican Representative Tom Cole, who sponsored the measure, said Congress had "allowed the president to overreach in Libya".
"We should not be engaged in military action of this level unless it is authorised and funded by Congress," he said.
'Wrong message'
However, Senator John McCain, also a Republican, said the vote was "deeply disturbing".
He said he shared frustration over President Obama's "lack of consultation" with Congress over the conflict but added: "This action sends the wrong message to both Gaddafi and those fighting for freedom and democracy in Libya - especially with Gaddafi clearly crumbling."
On a more positive note for President Obama, the House voted 229-199 against a measure that would have barred funds for US participation in the Libya mission.
The amendments were being debated as part of a $649bn (£406bn) defence spending bill.
Although the bill includes no funds for the Libyan operation, the Pentagon has said it can cover the costs with existing funds.
Last month, representatives voted overwhelmingly to deny President Obama authority to continue US participation in the operation in Libya, but rejected a bid to cut off money for the conflict.
Mr Obama says he does not need additional congressional approval, as US forces are simply supporting Nato.
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