"We are frustrated by the repeated unauthorized use of the song after a previous request to desist, which has obviously been ignored by Mr. Trump and his campaign," the band said in a statement issued by their publishing company, Sony/ATV Music Publishing.
"Queen does not want its music associated with any mainstream or political debate in any country. Nor does Queen want 'We are the Champions' to be used as an endorsement of Mr. Trump and the political views of the Republican Party. We trust, hope and expect that Mr. Trump and his campaign will respect these wishes moving forward."
A representative for Trump did not immediately respond to a request for comment.
During the first night of the Republican National Convention, Trump walked onstage to "We Are the Champions" before introducing his wife, Melania, who gave the night's headlining speech (which, coincidentally, also came under fire for allegedly incorporating someone else's work without permission). On Twitter, Queen responded to the spectacle, posting, "An unauthorised use at the Republican Convention against our wishes."
In June, Queen sparred with Trump over the use of "We Are the Champions," which was played before the candidate gave a victory speech after clinching the final round of Republican primaries. Guitarist Brian May wrote on his website, "This is not an official Queen statement, but I can confirm that permission to use the track was neither sought nor given. We are taking advice on what steps we can take to ensure this use does not continue. Regardless of our views on Mr. Trump's platform, it has always been against our policy to allow Queen music to be used as a political campaigning tool."