The outspoken star, who is best known for playing Pomona Sprout in "Harry Potter and the Chamber of Secrets", has hit out at the two funnymen, describing the rude treatment she experienced as a member of Cambridge University's Footlights drama club, which also spawned comedy legends Peter Cook, Stephen Fry, and Hugh Laurie.
In her new memoir "This Much Is True", out on 16 September (21), she writes, "I didn't like the Footlights boys and they really didn't like me. They made that obvious."
"When I say 'they,' I refer to a most distinguished group," she continues, naming Cleese and Chapman, as well as other British male comedians of that generation.
"(I was) the only girl in the show, I was a pert little madam and thought I was as good as they were - and they didn't."
She claimed she got the impression they didn't like her humour and suggested Python's shows "didn't feature funny women."
"My perception was that they thought I was a jumped-up, pushy, overconfident, fat little Jew. But I was funny, and they didn't like it."
"If you think about it, the Monty Python shows didn't feature funny women, only the occasional dolly bird (bimbo). And I certainly wasn't that."
And while Miriam hailed the comics as "men of genius," she still claimed they "were not gentlemen."
"I admire the creation of Monty Python and The Goodies (comedy trio) and I think they were men of genius, but they were not gentlemen."
"John Cleese, (The Goodies star) Bill Oddie and Graham Chapman were total s***s - and they have never apologised. The only one who did was the late (The Goodies regular) Tim Brooke-Taylor."
"All the perpetrators went into light entertainment and I went into drama, so thankfully our paths were seldom to cross," Margolyes adds.
"But nearly 60 years later I have not forgotten."
Graham Chapman, who played King Arthur in "Monty Python and the Holy Grail", passed away in 1989.
Brooke-Taylor died last year (20).