The Harry Potter star shared a scene with the Only Murders In The Building actor in 1986 comedy musical Little Shop Of Horrors and wrote in her new memoir Oh Miriam! that working with him had been a "vile experience".
Margolyes, 82, told news.com.au: "I just thought he was rather horrid. He was a c***, that’s all I can tell you."
She added: “I didn’t enjoy it and I had a splitting headache at the end of the day.
"He [Steve Martin] was incredibly unfriendly, because he was a perfectionist.
"He was an artist and all he was interested in was getting the comic moment right, and he was correct to do that, but he should have included me."
But the Call The Midwife star also called Martin a "gifted" genius.
She said: "Steve was and is brilliant, this is not about his talent, just about his kindness at that time in his life.
"When I saw the scene afterwards I thought, 'Oh, he’s good, he really is good'."
American comic Martin is not the only star who Margolyes has criticised of late.
The pair worked together in the early 1960s as part of Cambridge University’s renowned comedy society, the Cambridge Footlights.
Margolyes said of Cleese: “Like milk, he’s gone sour. He’s an irrelevance... Cleese is a puny tadpole of a person.”
She calls the Rolling Stones singer a “miserable c***” and the Terminator star “a very boring man”.
Margolyes began her acting career in theatre, film and TV playing supporting comedic roles, often much older than she was herself at the time.
She won a generation of younger fans when she was cast as Professor Sprout in the Harry Potter film series in 2002 and has since branched out into presenting travel documentaries, starting with The Real Marigold Hotel on BBC in 2016.
Watch: Miriam Margolyes swears on This Morning