Second-seeded Marat Safin of Russia was also beaten Wednesday in a day filled with huge upsets.
A seven-time champion, the sixth-seeded Sampras crashed out in the second round, falling to Switzerland's George Bastl, 6-3, 6-2, 4-6, 3-6, 6-4. The third-seeded Agassi, the 1992 champion and runner-up to Sampras in 1999, suffered a 6-4, 7-6 (7-5), 6-2 defeat at the hands of Thailand's Paradorn Srichaphan.
This is the first Wimbledon Championships in 12 years in which neither Sampras nor Agassi will reach the quarterfinals. In 1990, Sampras lost in the first round and Agassi did not enter the tournament.
Considered by many tennis' greatest grasscourt player in history, Sampras won a record seventh Wimbledon singles championship in 2000 but has not claimed a title anywhere since -- a drought of 30 tournaments.
He had won 56 of his previous 57 Wimbledon matches before losing to another Swiss player, Roger Federer, in the fourth round last year.
"This is more of a shock to me. As predictable as I've been over the years here, you're going to have a match like this once every 10 years, and it happened today," Sampras said. "It's going to be a tough flight home, a tough next couple of weeks, just knowing that this is going on and I'm not here."
The 30-year-old Sampras was especially downcast, considering Bastl has a lowly world ranking of 145 and got into the tournament because another player withdrew.
Bastl, 27, had not won at Wimbledon before his first-round victory Monday over Russia's Denis Golovanov.
"I gave myself chances because I had been practicing on grass for the last three weeks here in England and played a few warmup tournaments," Bastl said. "I knew coming into this match, I had won already three matches. So my confidence was good. I knew my game was improving match after match."
Sampras won his opening-round match in straight sets on Monday against Martin Lee despite having an injury described as an "acute strain in the lower right ribs."
Agassi lost to a player who never had advanced past the second round at the All-England Club. But Srichaphan, the No. 1 player in Thailand, reached his first career ATP final in January at the hardcourt event in Chennai, India.
"I'm a little stunned, certainly disappointed. I just never found my rhythm out there today," Agassi said. "I played a very average match against a guy who was taking it to me and deserved to win."
Srichaphan fired 15 aces and converted six break-point chances.
"He was playing aggressive and he was serving big. He was taking his chances," Agassi said. "This is a bit of shocker for me today. I felt good the whole time leading up to the tournament. It just shows you what you have to deal with out there every day. You're a little off, and that's enough to make the difference."
The 32-year-old Agassi is three years removed from becoming the fifth man to win all four Grand Slam titles in his career when he captured the French Open.
However, Sampras and Agassi have yet to lift a Grand Slam trophy in 2002 with just the U.S. Open left. It could be the first time in 11 years that neither has won at least one major tournament.
Meanwhile, Safin was eliminated by Belgium's Olivier Rochus, 6-2, 6-4, 3-6, 7-6 (7-1).
"Whatever I did, it was wrong. I just felt like I couldn't play," Safin said. "I was missing everything. It's frustrating. Unfortunately, it's a bad day at the office."
On the women's side, Americans Serena Williams and Jennifer Capriati eased into the third round.
Williams, the second seed, registered seven aces and 17 winners en route to a 6-3, 6-3 win over Italy's Francesca Schiavone.
"It was a little windy today and I don't think I was focused enough," the reigning French Open champion said. "Maybe my mind was in too many places. I just kept making a lot of errors. I didn't come up to the net nearly enough."
Capriati, the third seed, needed just 56 minutes to defeat Marta Marrero of Spain, 6-2, 6-1.
"I was able to be quite aggressive with my groundstrokes and I think I served pretty well," the two-time reigning Australian Open champion said. "I think your mind-set has to be totally different (on grass). I try to always look to be coming into the net and be more aggressive in taking the opportunity."
Four other men's seeds fell Wednesday.
Wayne Arthurs knocked off No. 12 Jiri Novak of the Czech Republic, 6-3, 7-6 (11-9), 6-3, and fellow Australian Mark Philippoussis overpowered No. 14 Thomas Enqvist of Sweden, 7-5, 6-3, 6-4.
A pair of American seeds also were eliminated. Arnaud Clement of France defeated No. 26 Todd Martin, 6-3, 6-4, 7-6 (7-2), and former Wimbledon champion Richard Krajicek of The Netherlands outlasted No. 29 James Blake, 6-3, 6-4, 3-6, 4-6, 11-9.
Meanwhile, fifth-seeded Russian Yevgeny Kafelnikov got past Nicolas Thomann of France, 7-6 (10-8), 6-2, 6-2, and 11th-seeded American Andy Roddick cruised to a 6-1, 6-2, 6-2 victory over Spaniard Alberto Martin.
Five women's seeds were ousted in the second round.
France's Mary Pierce defeated eighth-seeded compatriot Sandrine Testud, 6-3, 6-4, Mariam Oremans of the Netherlands knocked off 13th seed Meghann Shaughnessy of the United States, 7-5, 6-3, and American Chanda Rubin rolled over 14th seed Iroda Tulyaganova of Uzbekistan, 6-3, 6-1.
Maja Matevzic of Slovenia rallied past No. 22 Anne Kremer of Luxembourg, 2-6, 6-1, 6-2, and Els Callens of Belgium posted a 2-6, 6-3, 6-4 comeback win over No. 30 Clarisa Fernandez of Argentina.
However, No. 7 Jelena Dokic of Yugoslavia outlasted Kveta Hrdlickova of the Czech Republic, 6-0, 4-6, 8-6, No. 9 Amelie Mauresmo of France eased past Italy's Rita Grande, 6-4, 6-2, and No. 11 Daniela Hantuchova of Slovakia outdueled compatriot Martina Sucha, 6-2, 4-6, 6-3.
Also moving into the third round were Russians Anastasia Myskina and Tatiana Panova, the 18th and 21st seeds, respectively, 25th seed Nathalie Dechy of France and 26h seed Daja Bedanova of the Czech Republic.