Williams, 45, only returned to competitive tennis in July following a 16-month absence from the sport, and had been granted a wild card into the main draw at Flushing Meadows.
But the American tennis icon was unable to prolong her stay in the championship past the first hurdle, falling to a 6-3, 2-6, 6-1 defeat against her 29-year-old Czech opponent, who had not even been born when Williams turned professional in 1994.
Williams had not played a Grand Slam singles event since exiting in the first round of the US Open in 2023, and had not won a match at Grand Slam level since reaching the second round at Wimbledon in 2021.
Yet over the course of an entertaining battle on the Arthur Ashe Stadium, Williams offered glimpses of the formidable talent that earned her seven career Grand Slam singles titles, including five Wimbledon championships.
After starting slowly, suffering a break of serve in the opening game, Williams came storming back to level the first set at 2-2.
But Muchova pounced once more in the seventh game, Williams again struggling on serve to fall 4-3 behind.
Muchova held for 5-3 and then broke Williams again to take the set, the American veteran double-faulting on set point to groans from the crowd.
Yet any sense of a momentum shift in Muchova’s favour evaporated in the second set, Williams hitting back to break in the opening game before breaking Muchova again in the seventh game to open up a 5-2 lead and holding to force a deciding set.
Williams visibly ran out of steam in the third set, though, with Muchova securing an early break before holding for a 3-0 lead, and maintaining her advantage as she closed out victory. (AFP)