It was the sisters’ first loss as a doubles team at the Games, having won 15 previous matches together, taking the title in 2000, 2008 and 2012.
Just a day after 36-year-old Venus, suffering from a virus and stomach complaint, was beaten in the singles, the top-seeded sisters slumped to a 6-3, 6-4 defeat against the experienced Czech pair.
“It was what it was. We have a chance to compete for our country and did the best that we can. We had a blast out there,” said 34-year-old Serena.
“I wouldn’t say it was devastating. It was a lot of fun and we will always remember these moments and these matches.
“At the end of the day, I think that’s what matters most.”
She added: “So many Olympic doubles golds and Grand Slam titles, it’s been awesome.”
Venus said she and Serena will now focus on the US Open later this month.
“It was not our best day. We like to have a chance to play our way into the tournament,” said Venus.
“But we came up against a very strong team. We wish them the best of luck and we’ll get ready for the US Open.”
Serena is the defending champion in singles while Venus was the title winner in Sydney 16 years ago.
Serena is still in contention for back-to-back Olympic titles having defeated Australia’s Daria Gavrilova in her singles opener earlier Sunday.