Pegula, who reached the semi-finals at the last two Grand Slams, had her earliest exit in Paris since 2020, while it is also just the second time in six years that she has fallen at the first hurdle at a major. She was defeated by Elisabetta Cocciaretto at Wimbledon last year.
Pegula started her clay-court season in style, winning the title in Charleston before losing in the Madrid third round and Rome quarter-finals. She has never made it past the last eight stage at the French Open.
For Birrell, it is just the world number 83's third-ever win at a Grand Slam, and her first over a player ranked in the top five.
It is also the first time she has passed the opening round at a major since 2023, and she will take on Ukraine's Oleksandra Oliynykova next.