Saturday's "Bare with us" march took place in Waterloo, Ontario. The women say that police told them to cover up whilst cycling in the neighbouring town of Kitchener last month.
They have filed a formal complaint with the police.
It is legal for women to be topless in Ontario after a court ruling in 1996.
Protestors held signs that included the slogans "They are boobs not bombs, chill out" and "Nudity isn't sexual."
The three sisters, Tameera, Nadia and Alysha Mohamed, say that they took their shirts off because it was a hot summer day.
However, they allege that a police officer approached them and told them to cover up. But when they challenged this, the officer said he was stopping them for bike safety reasons.
One of the sisters is an award nominated Canadian singer under her stage name Alysha Brilla.
"I had no idea how polarizing the issue would be. I thought people wouldn't be so disturbed by the female breast," she told CBC news.
"We just want to advocate and let people know that they do have this right," the singer added.
Ontario passed legislation confirming the right of women to go topless in 1996, after the Ontario Court of Appeal overturned a woman's conviction for removing her shirt.
Gwen Jacobs had been fined in 1991, but on appeal the court found that there was "nothing degrading or dehumanising" about her going topless in public.
Hundreds of Canadian woman joined a topless protest march after three sisters were allegedly stopped by police for cycling without shirts.