Williams and Czech doubles partner Karolina Muchova fell to Mexico's Giuliana Olmos and Erin Routliffe, of New Zealand, 6-4, 6-4.
The duo lost the opening set after Williams was broken in her second service game.
They were broken on Muchova's serve midway through the second set and failed to find a way back into the contest.
The match was the 44-year-old Williams' second after her shock comeback announcement earlier in June.
She won her first match back at Queen's Club last Monday, but her tournament ended due to an injury to her doubles partner, Victoria Mboko.
The 23-time Grand Slam singles champion stepped away from tennis in 2022 but announced a comeback earlier in June, saying her decision came from a desire to play in front of her two young daughters.
Showing glimpses of the powerful game that allowed her to dominate women's tennis for two decades, Williams understandably lacked rhythm, particularly early, after a near four-year absence.
She got better as the match went on, improving her footwork and showing the powerful serve and trademark forehand, which were cornerstones of her game.
With one final singles wildcard slot remaining for Wimbledon, which starts on June 29th, speculation has grown that Williams could also make an individual return.
The last of her 23 Grand Slam victories came in the 2017 Australian Open. She last won Wimbledon in 2016.
The Williams sisters have won 14 Grand Slam doubles titles together, including six at Wimbledon.