The loss marks the third time Williams has come up short in Rio for her fifth career Olympic gold medal. But the second set was nearly a blowout, with Mattek-Sands and Sock dominating 6-1.
Bethanie Mattek-Sands, of the United States, and partner Jack Sock, right, hold their hand to their heart during the national anthem after winning their mixed doubles gold medal match against Venus Williams and Rajeev Ram.
After a back-and-forth first set that lasted 55 minutes, Mattek-Sands and Sock jumped to a 2-0 lead in the second set before Williams and Ram clawed back, thanks in part to Mattek-Sands, who suddenly lost her serve.
Mattek-Sands and Sock trailed 6-3 in the deciding super tiebreak then won six straight points.
The victory marked the second medal of the Rio Games for Sock. She is tied with Britain's Kathleen McKane, who won five medals between 1920-24.
Hradecka and Stepanek's win meant that the Czech Republic had finished off with a bronze medal in each of the three categories of the sport.
The rest of the world will be watching Usain Bolt race for an historic third Olympic gold medal in the men's 100m sprint.
The Russians got a break in the ninth game of each set to wrap up a 6-4, 6-4 win to claim the gold medal, while the Swiss team had to settle for the silver.
Venus won singles gold at Sydney in 2000 and doubles titles with sister Serena in 2000, 2008 and 2012.