A member of the public reported seeing a group kick the penguin in the middle of tram tracks at stop 136, Alfred Square/The Esplanade in St Kilda about 11.30pm on December 8.
The witness intervened, before relocating the penguin to the beach near St Kilda Pier.
The Conservation Regulator was quickly notified about the incident.
The regulator has released CCTV footage of three men they believe may be able to assist with their enquiries.
It’s illegal to injure, disturb, chase, or interfere with wildlife, with maximum penalties ranging from $3846 to $48,077 or 12 months in jail.
