In April last year, Grealish was cautioned about his conduct by Villa's former manager Tim Sherwood after photographs emerged of him allegedly inhaling the laughing gas in a hotel room.
"We instantly started an internal investigation about it, which is still ongoing. When it is finished we will see what disciplinary action to take," Di Matteo told British media.
"We expect our players to behave in a professional manner at all times. In general for the sake of any player they need to learn about what it takes to be an athlete."
Last season Grealish was banished to the under-21 squad after reports of a visit to a Manchester night club hours after a 4-0 loss at Everton in November, missing two Premier League games in a campaign which ended with Villa being relegated.
"It's important he (Grealish) understands what he has and that he needs to grow up very quickly," added former Chelsea player and manager Di Matteo. "It's not the first time and he needs to understand if he wants a good career he has to stop."
Grealish has scored twice in seven Championship appearances this season, with the club sitting 18th in the standings.
(Reporting by Ian Rodricks in Bengaluru; Editing by Ken Ferris)
