The Harry Potter star recently took her stage show Dickens' Women to Canada, and she received a rapturous response from the audience during a performance in Vancouver.
However, one woman in the front row remained seated during the standing ovation, prompting an angry reaction from Margolyes, who waited for the crowd to sit back down again before asking her why she didn't take part.
According to website Talkinbroadway.com, the unnamed theatre-goer claimed she had only ever taken part in one standing ovation and that was for a play featuring renowned British actor Sir Derek Jacobi.
Margolyes reportedly replied, "I'm sure Derek Jacobi deserved your standing ovation. So should Dickens. You're very rude."
The actress has now spoken out about the incident, revealing she feels bad for embarrassing the woman.
She tells the London Evening Standard newspaper, "It's true. I had no business to call her on it - but that's what I'm like."