Pliskova recovered from 2-1 down in the second set and briefly put Garcia on the backfoot, taking a 5-4 lead before the Frenchwoman saved two set points and held serve to force the tie-break, which she dominated.
It was the first time since 2008 that the top-seeded woman lost in the second round at the Open.
Mladenovic suffered a meltdown for the ages as she slid to defeat, collapsing from a 6-1, 5-1 lead and blowing four match points along the way.
Australian Davis Cup star Jordan Thompson is a win away from his career-best grand slam run after marching into the US Open third round for the first time. The Czech was getting to deuce, but couldn't generate a break chance as the Frenchwoman maintained her advantage.More news: Nvidia Benchmarks RTX 3080 Against RTX 2080 Ti In Doom Eternal
The match also marked the earliest exit by a top seed at Flushing Meadows since Simona Halep's opening round loss in 2018.
"I think she played (an) unbelievable set and a half", Pliskova told reporters. Another minibreak was achieved after Garcia's forehand was judged to have hit the line following a review.
Garcia will face American Jennifer Brady in the third round. Last year, she got knocked out in the first round. That was definitely one ball I could make. Speaking to the press after her loss in New York, Pliskova said, "No, zero pressure from this for me".
She managed to avoid a first-set shutout in picking up the sixth game. "Did you see the match or no?" and soon after remarked, "I don't know if you understand tennis well enough". "I mean, we just started". She was not going to give me the match for sure so I had to be ready for everything. "When I got broken in the second set I didn't do anything really bad, and I had to stay focused and aggressive".
Also Wednesday, Naomi Osaka, two-time Grad Slam victor and now passionate advocate for racial justice in America, returns to the court for a second-round matchup against Italy's lowly-ranked Camila Giorgi.More news: Immortals Fenyx Rising: Microsoft Store leaks details
Alexander Zverev (Ger) bt Brandon Nakashima (USA) 7-5 6-7 (8-10) 6-3 6-1, David Goffin (Bel) bt Lloyd Harris (Rsa) 7-6 (8-6) 4-6 6-1 6-4, Jan-Lennard Struff (Ger) bt Michael Mmoh (USA) 6-2 6-2 7-5. Giorgi made 24 unforced errors during the match.
"I was playing really good tennis there and I just couldn't close it out, couldn't convert my match points".
World number one Djokovic dropped the first against Britain's Kyle Edmund but rallied to progress 6-7 (5-7), 6-3, 6-4, 6-2 inside a subdued, spectator-free Arthur Ashe Stadium at Flushing Meadows.More news: North Korea vows 'severe' punishments in wake of typhoon