Their hopes of winning a fourth successive title were surprisingly ended in the first round.
The American top seeds lost 6-3 6-4 to Czech Republic’s Lucie Safarova and Barbora Strycova, who were only paired together at late notice.
Strycova replaced Karolina Pliskova, who withdrew because of Zika concerns.
This is the Williams’ first defeat in 16 Olympics matches playing together.
The sisters won gold together at Sydney 2000, Beijing 2008 and London 2012, but did not compete at Athens 2004 after Serena pulled out through injury.
“We played terrible and it showed in the results,” said 22-time Grand Slam singles title winner Serena.
Venus, 36, also lost in the singles on Saturday.
The U.S. women’s Olympic tennis coach Mary Joe Fernandez had said afterwards that the seven-time major champion had been sick since before she arrived in Brazil.
Serena, 34, can still become the first woman to win two Olympic singles titles after the London 2012 gold medallist beat Australia’s Daria Gavrilova earlier on Sunday.
But Venus, who won the individual title at Sydney 2000, saw her hopes of a fifth gold ended by Safarova and Strycova.
Neither of the unseeded Czech pair had previously won an Olympic doubles match, also losing their only previous outing together in a Fed Cup match last year.
“It was what it was,’’ Serena said. “We have a chance to compete for our country and did the best that we can. We had a blast out there.
“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.’’
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