Microbiologists Keith Warriner, who conducted the tests for the CBC's "Marketplace" program, said the discovery of clostridium difficile and methicillin-resistant staphylococcus aureus (MRSA) was "alarming."
"It was a surprise at the start, but amazing that all these hotels had superbugs," he said. "When you get ... the antibiotic-resistant bacteria we're finding, that's not scare-mongering, that's real. These are real pathogens that can cause real illnesses."
The U.S. Centers for Disease Control and Prevention says MRSA kills over 15,000 Americans each year.
Warriner examined rooms at EconoLodge, Super 8, Best Western, Holiday Inn, Fairmont and Sheraton rooms in Vancouver, Toronto and Montreal.
He was initially surprised by the presence of the superbugs in so many hotel rooms, he said.
"But if you think about lack of sanitation, multiple people coming in every day, it wasn't surprising in that respect."
Hotel chains reacted to the "Marketplace" broadcast.
"Seeing it on camera suggests that we need to revisit our housekeeping practices, specifically at these hotels," Tim Oldfield, EconoLodge's managing director of franchise performance said.
The findings, he said, didn't meet "my expectation of the standards we set as an organization."