Williams, 37, was due to face Zarina Diyas in Cincinnati on Tuesday, but pulled out as the American great continues to struggle with her back.
The 23-time grand slam champion retired during the Rogers Cup final against Bianca Andreescu on Sunday due to back spasms.
Two-time #CincyTennis champ Serena Williams has withdrawn."I came to Mason on Sunday and have tried everything to be ready to play tonight, and was still hopeful after my practice this morning. But unfortunately my back is still not right."Second night match: Pegula vs. Diyas pic.twitter.com/OGhmPnJgSY
— Western & Southern Open (@CincyTennis) August 13, 2019
"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," two-time champion Williams said, via the tournaments Twitter account.
"But unfortunately my back is still not right."
Williams withdrawal is a concern with the US Open less than two weeks away and the veteran still aiming to equal Margaret Courts record of 24 grand slam titles.
Jessica Pegula, the Citi Open champion, has taken Williams place and will face Diyas, who knocked her out in qualifying