Grealish reportedly attended a party where nitrous oxide - otherwise known as laughing gas - was used until the early hours of Sunday morning, and it prompted the club's owner Dr Tony Xia to message the winger on Twitter.
Ahead of the club's game with Newcastle United on Saturday, Di Matteo was quizzed over the alleged incident and he acknowledged that he had been left unimpressed with the reports that emerged from the weekend.
The 46-year-old told BBC Radio WM:: "Unfortunately it has happened again. We expect our players to behave in a professional manner at all times, so it is something that we don't tolerate.
"It's important that he understands what he has, and he needs to grow up very quickly."
Grealish played the full 90 minutes for Villa last weekend as they played out a goalless draw with Ipswich Town.