"I came to Mason on Sunday and have tried everything to be ready to play tonight, and was still hopeful after my practice this morning," the 23-times Grand Slam champion said.
"But unfortunately my back is still not right."
Williams, twice champion at the Cincinnati Masters, had been hoping to use the tournament as a final tune-up ahead of this month's U.S. Open.
She had been in menacing form in Toronto before retiring early in the first set of the final against Canadian teenager Bianca Andreescu. (Reporting by Rory Carroll; Editing by Peter Rutherford )
Chris Froome (Stage 3, 2015)
James Toseland (1/2)