The genus Fritillaria comprises seven species of the family Liliaceae and is an ornamental plant found in Kashmir at altitudes of 7000 to 9000 ft. Its bitter bulbs are traditionally used to treat sore throat, cough, asthma, bronchitis, scrofula, gland tumors, dysurea, and haemoptysis, and the genus is a rich source of steroidal bases. A new steroidal alkaloid, ebeinone (1), was isolated from the bulbs of Fritillaria imperialis along with four known steroidal bases—eduardine (2), edpetilidine (3), verticinone (4), and isoverticine (5)—which were obtained for the first time from this plant. The structures of these steroidal alkaloids were established through detailed spectroscopic studies. Compound 1 (ebeinone) at 1 pg/ml exhibited anticholinergic activity by completely blocking acetylcholine-induced contractions in isolated guinea pig ileum and atria, with no effect on histamine-induced contractions, indicating specific acetylcholine receptor blockade. Compounds 2–5 (up to 30 pg/ml) showed no inhibitory action against acetylcholine or histamine-induced contractions. The anticholinergic activity of ebeinone may underlie the traditional use of Fritillaria imperialis in treating asthma.