She was born in Oxford to a Jewish family originally from Belarus. She read English at Newnham College, Cambridge, and appeared in the Cambridge Footlights and later with Gay Sweatshop. She appeared in the TV series Blackadder and was "Pomona Sprout" in Harry Potter films. She played "Vera Sackcloth-Vest" in the radio series Gloomsbury.
Margolyes has been with her partner Heather for more than 40 years. She mentioned her relationships with women on several occasions when she appeared on BBC Radio 4's Desert Island Discs in September 2008. On becoming an Australian citizen, in 2013, Margolyes referred to herself as a "dyke" live on national television.
She was ranked number 93 in the Pink List 2006:
- "Margolyes, 66, would have been perfect as Charles Dickens's muse, seemingly made for roles in Oliver Twist, Little Dorrit and the 2002 biopic Dickens. Most recently Margolyes appeared in Stephen Hopkins's The Life and Death of Peter Sellers and Ladies in Lavender alongside fellow acting veterans, Dames Maggie Smith and Judi Dench."
- http://www.southernhighlandnews.com.au/news/local/news/entertainment/at-home-with-harry-potter-star-miriam-margolyes/1826576.aspx Leah O'Brien, "At home with Harry Potter star, Miriam Margolyes – Local News – News – Entertainment" Southern Highland News, 11 May 2010
- http://www.bbc.co.uk/radio4/factual/desertislanddiscs_20080928.shtml Desert Island Discs – 28 September 2008 – Miriam Margolyes. BBC. (28 September 2008).