Venus exit seals dismal day for the Williams sisters as their reign ends.
Defending champion Serena saw her bid to complete a hat-trick of titles brought to a crushing halt by French ninth seed Marion Bartoli, who won 6-3 7-6 (8/6) on Court One.
Venus, a five-time champion at the All England Club who last lifted the trophy in 2008, then lost for the second year running to Bulgarian number 32 seed Tsvetana Pironkova, 6-2 6-3.
"This was definitely not our best day. I think we both envisioned seeing this day going a little bit different," said Venus.
Wimbledon's top seed and world number one Caroline Wozniacki also bowed out on a day of big-name casualties, losing a nailbiting clash to Dominika Cibulkova 1-6 7-6 (7/5) 7-5. Today, bCibulkova will face 2004 champion and firm title favourite Maria Sharapova, who reached the quarter-finals with a 6-4 6-2 victory over Shuai Peng.
Bartoli's prize for her first career victory over Serena Williams is a last-16 meeting with Sabine Lisicki, who beat Petra Cetkovska 7-6 (7/3) 6-1. Pironkova will face Czech eighth seed Petra Kvitova, who breezed through with a 6-0 6-2 victory over Belgian Yanina Wickmayer.
And fourth seed Victoria Azarenka will go up against the world number 80 Tamira Paszek for a place in the semi-finals after brushing past Nadia Petrova 6-2 6-2. Austrian Paszek beat Ksenia Pervak of Russia 6-2 2-6 6-3.