The Manchester City and Manchester United stars - both integral players in previous tournaments for Southgate - became the most notable casualties after it was revealed James Maddison and Curtis Jones had both left the squad.
Burnley's James Trafford, Liverpool's Jarell Quansah and Everton's Jarrad Branthwaite also miss out.
Grealish's lack of form for Manchester City in the 2023/24 season likely contributed to his exclusion.
The winger managed just three goals in 36 appearances across all competitions. While he was a regular under Southgate previously, his recent struggles for form and fitness appear to have proved costly.
Maguire, meanwhile, has been a key player for England under Southgate in the past three major tournaments, often selected on merit rather than seemingly on his club form.
However, a calf injury has kept him sidelined since April, raising doubts about the centre-back's fitness.
Posting on social media shortly after the news broke, Maguire revealed his "devastation" at not making the squad:
"I am devastated not to have been selected to play for England at the Euros this summer.
"Despite my best efforts, I have not been able to overcome an injury to my calf. Maybe I pushed myself too hard, to try and make it. Simply, I am absolutely gutted.
"For me, representing England is the highest honour. It means everything to me. If I can’t help the team as a player, I will support them as a fan – along with the rest of the country. Go and win it boys.
"Next, I will return to the supervision of the Manchester United medical team in order to prepare for next season."
The defender made his senior debut in 2017 against Lithuania and was pivotal for England as they reached the semi-finals of the 2018 World Cup in Russia.
His form at Manchester United has been indifferent since then however, with fans and pundits often critical of his displays amid many high-profile errors.
Despite inconsistent club form, Southgate selected him for the World Cup in Qatar, where England reached the quarter-finals.
Maguire boasts 63 caps for England, while Grealish has featured 36 times.
The news means places for Aston Villa's Ezri Konsa, Manchester United's Kobbie Mainoo as well as Crystal Palace's Eberechi Eze and Adam Wharton.
Southgate has now officially finalised his squad for the Euros, with their first game taking place against Serbia on June 16th.
The Three Lions face Iceland in their final warm-up game at Wembley on Friday evening.
England's Euro 2024 squad:
Goalkeepers: Dean Henderson, Jordan Pickford, Aaron Ramsdale.
Defenders: Lewis Dunk, Joe Gomez, Marc Guehi, Ezri Konsa, Luke Shaw, John Stones, Kieran Trippier, Kyle Walker.
Midfielders: Trent Alexander-Arnold, Conor Gallagher, Kobbie Mainoo, Declan Rice, Adam Wharton.
Forwards: Jude Bellingham, Jarrod Bowen, Eberechi Eze, Phil Foden, Anthony Gordon, Harry Kane, Cole Palmer, Bukayo Saka, Ivan Toney, Ollie Watkins.