Walker earned his 82nd cap which put him level with Raheem Sterling in 16th place on the all-time Three Lions appearance list as FIFA’s third ranked team hosted the fifth ranked side.
But Walker had to come off injured in the 20th minute and he was replaced by Aston Villa debutant Ezri Konsa.
Gareth Southgate revealed post-match that the City defender would be assessed over the next few days.
Walker started the match as skipper while Stones ended the game with the armband, with the right-back watching the second half from the bench.
He’d earlier spared England’s blushes with an impressive clearance as the final man with Vinicius Junior firing dangerously goalwards after a mix-up at the back.
It was a feisty encounter but one of few chances, Stones going close for the hosts with a shot from just outside the box on the hour mark that went too high to trouble Bento.
With the game heading to a goalless draw, it was the five-time World Cup winners who broke the deadlock late on.
Vinicius Junior had a shot saved by Jordan Pickford on 80 minutes as he raced through on goal but substitute, 17-year-old Endrick, was there to fire home for the Green-Yellow from close-range.
England called for offside but the goal was given after a VAR check.
The loss represented England’s first defeat since the 2-1 World Cup quarter-final loss to France in Qatar in December 2023.
The Three Lions follow this game up with another home friendly encounter, this time against Belgium.
The game, with Jeremy Doku’s Red Devils, takes place on Tuesday at Wembley Stadium.
Meanwhile, Doku played the second 45 minutes of Belgium’s 0-0 friendly draw with Ireland.
Our flying winger replaced Leandro Trossard at half-time as part of an triple interval change by Domenico Tedesco at Dublin’s Aviva Stadium.
Belgium enjoyed the lion’s share of possession in the contest but John O’Shea’s side had the best chance.
Evan Ferguson missed a first half penalty with his spot-kick saved by Nottingham Forest keeper Matz Sels.
The game represented 21-year-old Doku’s 19th senior cap for the Red Devils since making his debut in September 2020 in a 2-0 UEFA Nations League away win in Denmark at FC Copenhagen’s Parken Stadium.
Doku could come up against teammates Walker, Foden and Stones on Tuesday with Belgium facing England at Wembley, kick-off 19:45 (UK).
Mateo Kovacic and Josko Gvardiol helped Croatia to victory over Tunisia on penalties in the semi-final of the 2024 ACUD Cup.
The friendly football tournament is taking place in Cairo, Egypt, bringing together four men’s national teams: Croatia, Egypt, New Zealand and Tunisia.
Gvardiol played the full 90 minutes at Cairo’s 30 June Stadium while Kovacic was on for 69 minutes as the sides drew 0-0.
The 2018 FIFA World Cup runners-up emerged 5-4 winners from the spot thanks to goals from Marco Pasalic, Mario Pasalic, Lovro Majer, Bruno Petkovic and Andrej Kramaric.
They will now face Egypt in the final on Tuesday, after they beat New Zealand 1-0 at the New Administrative Stadium in Cairo in the other semi-final.
