Police made the discovery after being called to the room by hotel workers.
Inside they found snakes ranging from 12 inches to four and a half feet long.
"The snakes were not being suitably cared for and were in distress. The anxious officers called the Society for the Prevention of Cruelty to Animals who attended and took 40 pythons into their care," cops said in a statement.
The reptiles were in plastic bins and tubs of all sizes and some were cramped for space.
The hotel manager said the couple who were keeping the snakes had just checked in but were not there when Canadian police arrived.
Cops in Brantford, Ontario confirm keeping pythons at home is illegal in the city.
All of the snakes were alive and are expected to be fine after being cared for properly.
Just 11 days ago two Canadian boys were crushed to death when a rock python escaped from its enclosure.