The Cumbrian property, which doubled as Uncle Monty's Cottage in the 1987 film, is due to be auctioned later this month.
An online appeal site has now been set up, with fans being urged to rally round to save a piece of "Britain’s cultural history".
Withnail And I followed actors Withnail (Richard E. Grant) and Marwood (Paul McGann) on a trip from Camden Town to Uncle Monty's (Richard Griffiths) countryside cottage.
The site states: "If we fail to raise the asking price, or are outbid, then all the funds raised will go to charity and [we] will be no worse off for having tried. But if we succeed, then we will have secured the future of Uncle Monty’s for years to come."
Sleddale Hall, near Shap, was mainly used for exterior shots and is being auctioned with a guide price of £145,000.