All looked well for the fifth seed as she took the first set 6-4, but Williams let a 5-1 lead slip in the second-set tiebreak as Razzano claimed six-straight points to square things up.
The lapse continued for Williams as Razzano ran out to a 5-0 lead in the decisive third set. But as Razzano, ranked 111 in the world, began to show signs of cramps between points, Williams struck back to reel off three-consecutive games.
Then came an epic and ultimately decisive ninth game. Razzano was controversially given her second point penalty for excessive grunting, invoking a skeptical reaction from Razzano, and a furious response from the crowd that ratcheted up the drama to melting point.
Both players looked increasingly fraught with tense emotions, as break points and match points came and went in a game lasting 25 minutes. But on her eighth match point, Razzano finally claimed an emotional victory as the umpire confirmed that Williams' ball had landed just long.
While it is the exit of Williams, seen as a strong contender for her second French Open title, that will make the headlines, it is a heart-warming tale for her conqueror. Razzano suffered the loss of her fiancé, Stephane Vidal, last year from a brain tumor before just over a week later taking to the court at Roland Garros sporting a black ribbon. Though she lost in the opening round in 2011, her win on Tuesday proved a fitting tribute in what is arguably one of the biggest upsets in recent French Open history.
The result is certainly by far the biggest upset at Roland Garros this year with Williams widely fancied in Paris after winning the title in Madrid before having to withdraw from her semi-final in Rome earlier this month.
I made so many errors today, which isn't the game I was playing in the past, Williams said, according to ESPN. That's life.
The exit of the American will come as a boost to fellow title hopeful, Maria Sharapova. The Russian went onto claim the crown in Rome just over a week ago and will now feel she has arguably never had a better opportunity to claim her maiden French Open.
In sharp contrast to Williams, Sharapova raced through her opening round on Tuesday, defeating Alexandra Cadantu without the loss of a game.