Williams, 38, won three of eight breakpoints and had 38 unforced errors in the two-hour, 38-minute clash. Sakkari had 43 unforced errors in the loss. Williams won the three-set match 6-3, 6-7(6), 6-3 at Arthur Ashe Stadium.
Sakkari beat Williams in three sets in the round of 16 on Aug. 25 at the Western & Southern Open, which was played a week before the U.S. Open and also took place at the USTA Billie Jean King National Tennis Center.
"I felt like she almost played better today," Williams said of Sakkari. "She's such a good competitor so it was a really intense match."
Williams will face Bulgaria's Tsvetana Pironkova in the quarterfinals in her quest for a record-tying 24th Grand Slam. The winner of that match will play for a spot in the women's singles final.
Pironkova advanced to the quarterfinals after a three-set win over Alize Cornet Monday in Queens.
Dominic Thiem is the highest-seeded player in the men's singles circuit. He dispatched of Canadian Felix Auger Aliassime 7-6, 6-1, 6-1 to advance to the quarterfinals. No. 3 Daniil Medvedev also beat American Frances Tiafoe in straight sets to advance. No. 10 Andrey Rublev beat No. 6 Matteo Berrettini in four sets to make it into the quarterfinals.
Alex de Minaur also beat Canadian Vasek Pospisil in straight sets and will face Thiem in the quarterfinals. The winner of that match will face Rublev or Medvedev in the semifinals.
No. 4 Naomi Osaka will face American Shelby Rogers in a quarterfinal clash at 7 p.m. EDT Tuesday in Queens. Jennifer Brady will take on Yulia Putintseva in Tuesday's other women's singles quarterfinal. No. 5 Alexander Zverev faces Borna Coric at 1:15 p.m. EDT Tuesday in Queens in the first men's quarterfinal. Denis Shapovalov battles Pablo Carreno Busta in the other men's quarterfinal Tuesday in Queens.