The 23-time Grand Slam champion was half of the headline match of the tournament as she was set to square off with long-time rival Maria Sharapova.
The Russian, who has not beaten Williams since the 2004 WTA Championships, receives a walkover into the quarterfinals where she will face either third seed and 2016 Roland Garros winner Garbiñe Muguruza or unseeded Ukranian Lesia Tsurenko.
Williams picks up injury, unable to serve
Having just returned to Grand Slam action for the first time since winning last year's Australian Open, the American held a press conference and said: "I've unfortunately been having issues with my pec muscle and unfortunately it's been getting worse. Right now, I can’t actually serve so it’s actually hard to play when I can’t physically serve".
She indicated the injury was a new one, which was evident in her doubles match with sister Venus yesterday:"It didn't start before I got here. The first time I felt it was against Goerges in my last match.
"That's when I started to feel it. It was really painful and I didn't know what it was."
Three-time Roland Garros champion used doubles to see extent of injury
Serena and Venus accepted a wild card into the doubles draw, reaching the third round before losing in three sets to the third-seeded team of Andrea Klepac and Maria Jose Martinez Sanchez.
"In my doubles yesterday, I tried a lot of different tapings and support to see how it would feel under match circumstances. I'm not serving that often; I'm serving once every four games...I thought it was a perfect opportunity to see how I was," said Williams.
She added: "It didn’t get a lot better."
She also expressed her disappointment at not being able to take the court against Sharapova, who she has defeated 18 consecutive times: “It’s very difficult because I love playing Maria," she stated.
"It’s a match I always get up for and her game matches up against mine."
Serena to undergo MRI next Tuesday; Sharapova comments on withdrawal
Williams added that she: "made a promise to myself and to my coach and to my team that if I'm not at least 60% or 50%, then I probably shouldn't play".
She knew that:"the fact that I physically can't serve at all is a good indication that maybe I should just go back to the drawing board and stay positive and try to get better and not get it to a point where it could be a lot worse."
She will undergo an MRI tomorrow and won't speculate on any future plans until the results of that are known.
Williams said: "I'm going to stay here and see some of the doctors here, see as many specialists as I can. I won't know [about Wimbledon] until I get those results," she said.
"I just always try to think positive and just think of the bigger picture and hopefully the next events and the rest of the year."
For her part, Sharapova stated:"I was looking forward to my match against Serena today and am disappointed that she had to withdraw.
"I wish her a speedy recovery and hope she returns to the tour soon."