Running in doubles with partner Hayley Baptist at the DC Open, he finished in the second round of Wednesday, with a 6-4, 3-6, 10-6 loss on the champion tiebreaker to second seeded Taylor Townsend and Zhang Shuai.
Williams, 45, has made her first appearance in the tournament in over a year and was her first appearance in doubles since 2022, with her and Baptist winning their first match on Monday.
Williams will have a second round single match against Magdalena Fletch on Thursday.
On Tuesday, Williams became the second oldest woman to win a tour-level singles match, beating Payton Stearns, 23, 6-3, 6-4 in straight sets.
Following that victory – she was the first in nearly two years – Williams was awarded a wildcard entry on Wednesday at the Cincinnati Open next month.
Other DC Open Women’s Singles Play Wednesday, seedless Anna Karinkaya, No. Eliminate 8-seeded Magdalinette 6-4 and 6-0.
The Associated Press contributed to this report.