Williams won singles and doubles gold at the previous Games in London, four years ago. But the American has suffered a shock in the third round and leaves the Rio Games empty-handed.
World number one Serena Williams has been knocked out of the women's singles tournament in Rio after a 6-4, 6-3 defeat to Ukraine's Elina Svitolina.
Williams, who won her sixth Wimbledon championship this summer, is denied the opportunity to win her fifth medal at the Olympic Games. Serena and Venus exited the doubles tournament in the first round, losing to Czech pair Lucie Safarova and Barbora Strycova.
The 34-year-old was broken in the first set and couldn't recover against the Ukrainian. Williams appeared to be playing on with a shoulder problem and she was left in tears at the end of play.
"It's an unreal feeling for me. This is just a perfect moment," said Svitolina, who is ranked 20 in the world. The 21-year-old's personal best in a Grand Slam event is a quarterfinal finish at the French Open last year.
"I didn't really think about my record against Serena," she added. "I didn't expect that I could win and I said don't think she will make a mistake. Just fight and focus."
Williams refused to answer questions from the media initially, but told the U.S. Tennis Association that she felt the better player won in Rio. "It didn't work out the way I wanted it to, but at least I was able to make it to Rio. That was one of my goals," added Williams.