It is expected that many stations will have to close when the charges come into force on 16 July.
The US District Court of Appeals for the District of Columbia decided not to grant a stay of payment pending court deliberation.
The ruling means that the combined bill for royalties to be paid by US internet radio stations is more than $1bn, according to the SaveNetRadio coalition, a pressure group formed to oppose the charges.
SaveNetRadio believes that the online stations have made less than $200m between them.
The US Copyright Royalty Board ruled earlier this year that royalties for internet radio stations should rise from .08 cents per track to .19 cents by 2010, sparking a wave of protests.
In the wake of the court defeat, SaveNetRadio is urging its supporters to lobby congressmen to back a new bill, the Internet Radio Equality Act, which would bring internet fees in line with satellite radio stations.
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