The Three Lions captain picked up an ankle injury during Bayern Munich's most recent Bundesliga clash with SV Darmstadt 98, although he still linked up with the squad at St George's Park.
Little is known about the severity of Kane's issue, although he was one of a handful of players to miss team training on Wednesday, instead undergoing individual work in the gym.
The 30-year-old's problem has also poured doubt on his availability for Bayern's Champions League quarter-final with Arsenal next month, and Southgate's latest admission will not be music to the ears of Thomas Tuchel.
Speaking to the media in his pre-game press conference on Friday evening, Southgate announced that Kane would not be fit to take on Brazil, and nor would Chelsea's Cole Palmer and Ajax's Jordan Henderson.
Both men also sat out team training this week alongside Bukayo Saka, who withdrew from the squad on Thursday, but Southgate is keeping his fingers crossed that Palmer and Henderson can make themselves available for the meeting with Belgium next Tuesday.
"Tomorrow, no Harry Kane, no Jordan Henderson, no Cole Palmer," Southgate told reporters. "I would say Cole and Hendo have a better chance of Belgium than Harry, he'd be extremely doubtful for that."
With England's all-time record goalscorer unavailable, Southgate must choose between Aston Villa's Ollie Watkins and Brentford's Ivan Toney to lead the line, while Jarrod Bowen or Phil Foden could benefit from Saka and Palmer's absence to start on the right.
Alternatively, Foden could vie for Henderson's spot in midfield, although Conor Gallagher and the uncapped Kobbie Mainoo are also candidates to join engine room shoo-ins Declan Rice and Jude Bellingham.
Given the array of youthful talent at his disposal, Southgate is excited to see how England's fringe players step up in the absence of more revered names, adding: "It's a brilliant opportunity for other players.
"We need to see other players ahead of the Euros. If we were playing lesser teams you'd still have that little question in your mind; if they played well, OK, how are they going to be against the next level of opposition? From our perspective, it's a great exercise and a great opportunity for them."
While England have a handful of injury concerns to work around for Saturday's game, Brazil head coach Dorival Junior - who will take charge of the Selecao for the first time this weekend - has been hit by five major withdrawals.
Saka's fellow Gunners Gabriel Martinelli and Gabriel Magalhaes have both left the camp with injuries, while Manchester City's Ederson, Manchester United's Casemiro and Paris Saint-Germain's Marquinhos pulled out as well.
The likes of Neymar, Gabriel Jesus, Eder Militao and Alisson Becker are absent too, and the South American giants - who have lost their last three games - will field one of three uncapped goalkeepers in Bento, Leo Jardim or Rafael at Wembley.