Jodie Burrage knocked out Spanish top seed Paula Badosa and Katie Boulter defeated 2021 Wimbledon finalist Karolina Pliskova on a day of British success at Eastbourne.
Ryan Peniston beat another notable name on grass, fighting back to beat French Open quarter-finalist Holger Rune.
Meanwhile Dan Evans overcame France's Adrian Mannarino in straight sets.
Harriet Dart's meeting with Swiss player Jil Teichmann was suspended with the match level at one-set-all.
Dart, the second highest-ranked British woman behind the absent Emma Raducanu, won the first set but ended Tuesday's play tied at 7-6 (9-7) 4-6.
In the final event before Wimbledon, a jubilant Burrage celebrated a memorable win over world number four Badosa to reach the last 16.
The 23-year-old world 169 recorded a 6-4 6-3 victory over the Spaniard to seal by far the biggest win of her career, by ranking, to date.
She will play Brazil's Beatriz Haddad Maia, who extended her winning streak on grass to 11 matches with victory over Kaia Kanepi, for a place in the quarter-finals.
Likewise, Boulter secured her first win over a top-10 opponent as she came from behind to beat Czech Pliskova.
The 25-year-old British women's number four responded impressively after the Czech dominated the opening set to prevail 1-6 6-4 6-4.
"I'm super proud of myself today," said Boulter, who will play Czech two-time Wimbledon champion Petra Kvitova next.
"I worked so hard today to battle and fight. Against a player like that, it means so much to come through."
In the men's draw, Peniston kickstarted the day of British success with his 4-6 7-6 (7-5) 6-1 win over Denmark's eighth seed Rune.
The British men's number six, who beat world number five Casper Ruud on his way to reaching the Queen's quarter-finals last week, will now meet Spain's Pedro Martinez.
Rune was twice two points away from victory in the match - at 6-5 in the second set and 5-5 in the tie-break - but after being broken early in the third set, the 19-year-old drew jeers from the Eastbourne crowd for hitting a ball out of court and kicking his towel bin.
"I'm very happy with that. A tough start but I managed to fight thanks to you guys," Peniston told the home crowd.
"Since Queen's it has been madness. A couple of weeks ago was a lot different and things have changed. But I'm loving it."
British men's number two Evans also won his first-round match in the ATP event, earning a 6-4 6-3 victory over Mannarino.
Clinical breaks of serve in the 10th game of the first set and eighth game of the second sealed victory for world number 33 Evans, with his reward a second-round match against American Maxime Cressy.
The headline act at Eastbourne on Tuesday was, however, the return of Serena Williams to the court after almost a year out.
The 40-year-old 23-time Grand Slam singles champion, who had not played since she was injured at Wimbledon last year, won her women's doubles opener alongside Tunisia's Ons Jabeur 2-6 6-3 13-11 against Spain's Sara Sorribes Tormo and Marie Bouzkova of the Czech Republic.
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