Kerber lost 6-4 1-6 7-6(11) to Russian Ekaterina Makarova, while Williams was upset 6-3 2-6 6-2 by Australian qualifier Ashleigh Barty in second-round matches.
Barty will face former world number one Caroline Wozniacki, a 6-2 6-4 winner against Russia's Elena Vesnina, in the last 16.
Top seed Pliskova rose to the occasion on the key points.
She saved all five break points she faced, while losing just three points behind her first serve against the Russian, who reached her first WTA final at Den Bosch in June.
Last year Pliskova beat Kerber in the Cincinnati final, stopping the German from ascending to number one with the biggest victory of her career up to then.
Pliskova went on to have a brilliant Flushing Meadows run, beating both Venus and Serena Williams on the way to the final, sparking a consistent stretch that helped her succeed Kerber atop the rankings after Wimbledon.
Next up for the 25-year-old Czech is a third-round match against Italian Camila Giorgi, who beat Australian Daria Gavrilova 7-6(1) 5-7 6-3.
American 16th seed Madison Keys beat Russian Daria Kasatkina 6-2 6-1 to reach the last 16, where she will face Wimbledon champion Garbine Muguruza of Spain.
For the second week in a row, American Sloane Stephens defeated 14th seed Petra Kvitova, who was bothered by pain in her right thigh, to reach the third round with a 6-2 6-3 win.
Stephens, sidelined almost a year due to a foot injury before returning at Wimbledon, beat Kvitova last week in three sets in Toronto on her way to the semi-finals.
Up next for Stephens will be the on-form Makarova.
(Reporting by Frank Pingue in Toronto and Larry Fine in Tampa; Editing by Toby Davis/Peter Rutherford)
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