The Queensland bowling allrounder made a mess of the tourists as they crumbled to 5-64 at lunch, after Australia A captain Nathan McSweeney won the toss and elected to bowl first.
But Neser (4-27) managed to bowl just two balls of his 13th over, before pulling up sore and walking dejectedly off the field only minutes before the tea break.
Neser won't bowl again for the rest of the match and will undergo scans on his left hamstring to determine the damage.
India A were bowled out for 161, almost entirely thanks to a brilliant unbeaten 80 from wicketkeeper Dhruv Jurel.
Earlier, they collapsed to 4-11 during a chaotic first 16 balls of the match.
Neser was on a hat-trick in his first over after dismissing Abhimanyu Easwaran and Sai Sudharsan in consecutive balls.
Ruturaj Gaikwad survived the hat-trick ball, but fell in Neser's next over.
Michael Neser is congratulated by teammates as he swept through the India A top order. (James Ross/AAP PHOTOS)
In between, Scott Boland removed KL Rahul, an option to play against Australia in the opening Test of the Border-Gavaskar series, for just four.
The wickets tumbled so quickly that the scoreboard at the MCG failed to realise Rahul had been promoted to open after he had initially been listed to bat at No.4.
Rahul was dropped for India's third Test defeat against New Zealand last week, arriving in Australia early to try and find some form for India A.
Boland spoke about Rahul on Tuesday: "He's obviously a world-class player, but he is someone we can hopefully get on top of pretty early and stay on top of him for most of the summer."
It only took three balls from Boland (1-22) - and four overall - to get Rahul out at the MCG.
Neser had done his Test chances a world of good before his injury. (James Ross/AAP PHOTOS)
Boland hasn't played for Australia since the 2023 Ashes, but will be in consideration for a call-up this summer should pace stars Pat Cummins, Mitchell Starc and Josh Hazlewood be unavailable.
McSweeney bowled first for a second-straight match after Australia A won in Mackay last week.
The South Australian captain has been promoted to open the batting in this match as he closes in on a Test debut.
McSweeney fired with an unbeaten 88 in Mackay, putting him ahead of Marcus Harris, Cameron Bancroft and Sam Konstas in the battle to claim the vacant spot in the Australian XI.
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