The 39-year-old Williams, a three-time winner at Roland Garros, had arrived in Paris carrying an Achilles injury that prompted her to skip the Rome tune-up event.
“The Achilles didn’t have enough time to heal after the US Open,” said Williams.
“I was struggling to walk and that is a tell-tale sign that I should try to recover.”
The injury likely means she will miss the rest of 2020 leaving the Australian Open in 2021 as her next chance to equal Margaret Court’s all-time majors record.
“I need four to six weeks of sitting and doing nothing,” she said. “It’s more than likely that I won’t play another tournament this year.”
Her decision to pull out handed Bulgaria’s Tsvetana Pironkova a walkover and left Williams searching for a first major since the last of her 23 titles at Melbourne in 2017.
Later on Wednesday, Rafael Nadal looks to build on a promising start to his quest to match Roger Federer’s 20 Slam titles when he plays American outsider Mackenzie McDonald.
Top seed and women’s favourite Simona Halep also puts her 15-match winning streak on the line as she targets another run at a second French Open crown in three years.
Nadal needs one more major to pull level with long-time rival Federer and owns an astonishing 94-2 record in Paris going back to his triumph on debut in 2005.
The Spaniard showed little trouble in handling the heavier conditions in his opening win over Egor Gerasimov at a tournament postponed four months by the coronavirus pandemic.
However, the 34-year-old is wary of an untimely slip-up having made serene progress on his return to action in Rome before running aground in the quarter-finals.