Demonstrators chanting "we believe the women" in reference to the sexual abuse allegations levelled against the embattled comedian stood up shortly after Cosby launched into his comedic routine in Hamilton, Ont., waved signs, blew whistles and walked out of his performance.
The protesters, made up of a mix of men and women who had been seated at various points in the theatre, were escorted out by police. One man called Cosby a piece of excrement as he left the venue.
As the audience started to react, Cosby asked them to keep their cool.
"Stop. Stop," he said. "They've had their say and we can calmly let them out. Now, if I may."
Cosby was met with a round of applause before he went on with his routine.
The walk-out during Friday's show came a day after a man at Cosby's performance in London, Ont., called the comedian "a rapist."
It also took place just minutes after Cosby acknowledged the controversy that has followed him throughout his time in southwestern Ontario.
"Whatever happens here tonight, if there's some sort of outburst, we just have to remain calm and things will be taken care of. It oughtn't last that long," he said.
"I want to thank all of you here tonight because you stayed with what you believed in."
Cosby also acknowledged the protesters who have borne freezing temperatures to voice their opposition to him at every performance.
"They stayed with what they believed in," he said. "God willing, two years from now, when I come back, all the people outside will also be inside."
Cosby is facing growing sexual assault allegations, with some claims dating back decades. He has denied the allegations through his lawyer and has never been charged.
At the end of Friday's performance, which had been punctuated with frequent laughter and applause from the audience, Cosby made another reference to those who have been voicing their opposition against him.
"Let us take what we had tonight as a wonderful time and we hold no enemies anywhere. You maintained your calm," he said.
"Two years from now we may all be in this hall together to celebrate the gift that is laughter. Thank you."
Cosby's parting words were greeted with a standing ovation — his third in three consecutive nights.
"He was so professional and so polite," ticketholder Hilary Rose said of the way Cosby had handled the disruption to his show. " I'm glad I spent the money to come and got the opportunity to see him live."
Some in the audience, however, voiced disappointment in the demonstrators.
"It was embarrassing. It was terrible. I expected better," said Wendy Burn. "He's innocent until proven guilty. He's entitled to make a living, same as any other innocent person."
Those who had gathered outside the theatre earlier in the night to send a message to Cosby felt differently.
"We need to listen to the survivors of violence," said Kathryn Baker-Reed.
"While the allegations against Bill Cosby have certainly not been proven in a court of law, these women have been trying to have their voices heard for a long time. This a way for us as community members to say that we're listening, we're paying attention and we're really hoping that due process is provided."