Williams squandered a lead in each set Wednesday and lost her third-round match to No. 7-seeded Angelique Kerber of Germany, 7-6 (5), 7-6 (5).
Williams, seeking a record fourth gold medal in Olympic tennis, still has a shot with her sister Serena in doubles, where they're in the quarter-finals.
Venus looked sharp in the first two rounds of singles, as if rejuvenated by the setting, where she has won five of her seven Grand Slam titles. But she was diagnosed in 2011 with an autoimmune disease that causes fatigue, and looked weary against Kerber.
In the first set Williams failed to convert three set points, then blew a 5-1 lead in the tiebreaker. She was up 3-1 in the second set, but Kerber again charged back.
Kerber, a semifinalist at Wimbledon last month and at the U.S. Open last year, is ranked a career-high No. 7. She advanced to the quarter-finals against top-seeded Victoria Azarenka of Belarus.
Also reaching the final eight was Serena Williams, who hit 12 aces and repeatedly rocketed her groundstrokes past No. 13-seeded Vera Zvonareva to win 6-1, 6-0.
Swinging lustily with almost every shot, No. 4-seeded Serena hit 32 winners to three for the Russian, who also lost when they met in the 2010 Wimbledon final. The younger Williams swept the final 10 games and was done in only 51 minutes.
"I was just playing unbelievable," Williams said. "I was nervous going into the match and I didn't speak to anyone and I had a bad practice. I had no idea I would play like this."
Roger Federer endured two rain delays and a shaky moment late in the first set to reach the quarter-finals by beating Denis Istomin of Uzbekistan 7-5, 6-3.
Serving at 5-all in the first set, the No. 1-seeded Federer faced three break points and erased them all. He then broke and was in control from there.
A four-time Olympian, Federer has yet to win a singles medal, although he and Swiss teammate Stanislas Wawrinka did win the gold in doubles in 2008. Federer is playing his first tournament since winning a record-tying seventh Wimbledon title.
No. 2 Novak Djokovic hit 16 aces and came from behind to beat Lleyton Hewitt of Australia 4-6, 7-5, 6-1. Djokovic next plays No. 5 Jo-Wilfried Tsonga of France, who beat Feliciano Lopez of Spain 7-6 (5), 6-4 less than 24 hours after winning a 48-game third set.
Four-time Grand Slam champion Kim Clijsters, playing in her first Olympics weeks before she retires, beat former No. 1 Ana Ivanovic of Serbia 6-3, 6-4. Azarenka edged No. 16 Nadia Petrova of Russia, 7-6 (6), 6-4.
While play was interrupted by rain on other courts, Serena Williams worked under Wimbledon's roof, and the conditions clearly suited her. She whacked winners even from behind the baseline, and more than once spun on one foot after smacking a shot, mixing a little body English with plenty of brute force.
Williams hit a Wimbledon record 102 aces en route to her fifth title at the All England Club last month, and the shot remains a dominant force, especially on grass. She hit three consecutive aces against Zvonareva and lost only seven points in six service games.
In the second set, the crowd tried to coax a comeback from the Russian. But when she began tossing her racket in frustration, cheers for her turned to jeers.
And fans applauded Williams' domination.
"We love you, Serena!" a spectator near the top of the stadium shouted during the final changeover. Williams continued to stare sternly, but she wore a grin moments later as she walked off the court in triumph, waving to the stands dotted with U.S. flags.
The younger Williams has lost only 10 games through three rounds. The three-time Olympian is the winner of two gold medals in doubles and 14 major titles, but she has yet to win a singles medal.