Police found the snakes in five plastic storage bins at the Bell City Motel in Brantford, which is located about 100 kilometres southwest of Toronto.
"Early (Thursday) evening, the Brantford police called us to come out and assist with a large number of snakes they had found," Brandon James, spokesman for the Brant County Society for the Prevention of Cruelty to Animals (SPCA), was quoted as saying by CBC News.
He said that he had "never seen a load of snakes this large in Brant County and I've been doing this for 10 years."
The biggest python was estimated to be 1.3 metres long.
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There were snakes of various sizes including some that were less than a foot long. There was also about half a dozen unhatched eggs in the bins.
The pythons were discovered Thursday night by police as part of a separate investigation.
The snakes were in distress because of the cramped conditions and lack of water, James said.
James said it appeared the snakes had recently been fed.
The snakes belonged to a local couple who stayed at the room for only one night, a manager at the motel told The Canadian Press.
The couple were out in the evening when police arrived, said the manager.
Paul Sizer, a guest in a neighbouring room at the motel, told CBC News that the couple had five young children with them, including a baby.
"The first night I was afraid of (the snakes) getting out and coming after me," he said.
The discovery came after two boys were killed by an African rock python in New Brunswick earlier this month.