Good Morning to You Productions (GMTY), which is making a documentary on the tune, said it was forced to pay Warner/Chappell $1,500 to use the song in order to avoid being fined $150,000 for unauthorized use.
It has filed suit in a federal court in New York demanding that the government declare invalid the copyright over the song.
The the 26-page lawsuit also demands the return of "millions of dollars of unlawful licensing fees collected by defendant Warner/Chappell pursuant to its wrongful assertion of copyright ownership of the song."
"Defendant Warner/Chappell has collected and continues to collect at least $2 million per year in licensing fees for Happy Birthday to You," it added.
A spokesman for Warner/Chappell declined to comment.
According to GMTY, the tune comes from a song called "Good Morning to All," which was composed in 1893 by sisters Mildred and Patty Hill, who sold the rights to Clayton F. Summy.
The song in its present form was released in 1924 by Robert Coleman, setting off a series of legal disputes.
"Irrefutable documentary evidence, some dating back to 1893, shows that the copyright to 'Happy Birthday to You,' if there ever was a valid copyright to any part of the song, expired no later than 1921," the lawsuit says.
"If defendant Warner/Chappell owns any rights to Happy Birthday to You, those rights are limited to the extremely narrow right to reproduce and distribute a specific piano arrangement for the song published in 1935."
Mark Rifkin, one of the lawyers representing GMTY, told AFP Warner/Chappell has 30 days from when it receives the complaint to respond, with the option of asking a judge to dismiss the suit for the lack of merit.