The 23-time Grand Slam singles champion returned to tennis after almost a year out with injury at Devonshire Park this week, teaming up with world number 3 Ons Jabeur in doubles.
The duo beat Sara Sorribes Tormo and Marie Bouzkova in the round of 16 on Tuesday before booking a place in the semi-finals yesterday by ousting Shuko Aoyama and Chan Hao-ching.
They were due to face Magda Linette and Aleksandra Krunic in the last four on Friday, only to withdraw from the tournament this morning after Jabeur injured his right knee.
Williams will now prepare for her return to Wimbledon, where last year she suffered a serious hamstring injury in the first set of her first round match against Aliaksandra Sasnovich.
The American received a wildcard in the singles main draw at SW19, with her world ranking dropping to 1,204 in her nearly 12 months away from the court.
It is unclear at this stage whether Tunisia’s third seed Jabeur is likely to miss Wimbledon, which starts at the All England Club on Monday (June 27th).