Returning Pironkova stuns 10th seed Muguruza at US Open
Pironkova, who made a comeback to tennis this year after a three-year break following the birth of her son, stunned two-time Grand Slam singles winner Muguruza 7-5, 6-3 in 1hr 25min.
Pironkova, who does not have a world ranking due to her long absence from the circuit, will now face Croatian 18th seed Donna Vekic in the third round.
Vekic advanced to the last 32 on Thursday with a 6-2, 6-1 defeat of Romania's Patricia Maria Tig.
Prior to this week, Pironkova, 32, had not played in a Grand Slam or any event since her appearance at Wimbledon in 2017, when she lost in the second round of a tournament eventually won by Muguruza.
Thursday's victory marks only the second time that Pironkova has reached the third round of the US Open in 12 appearances stretching back to 2005.
Pironkova's best performance in a Grand Slam came in 2010, when she reached the semi-finals at Wimbledon.