The in vivo antifungal activity of crude extracts of Dipsacus asper roots was evaluated against the phytopathogenic fungi Botrytis cinerea, Colletotrichum coccodes, Blumeria graminis f. sp. hordei, Magnaporthe grisea, Phytophthora infestans, Puccinia recondita and Rhizoctonia solani using a whole-plant assay method. Ethyl acetate and acetone extracts, at 1000μg/mL, suppressed the development of tomato gray mold (TGM) and tomato late blight (TLB) by 90%. Through bioassay-guided isolation, five antifungal substances were isolated from the D. asper roots and identified as β-sitosterol (1), campesterol (2), stigmasterol (3), cauloside A (4) and a novel dipsacus saponin, named colchiside (3-O-β-d-xylopyranosyl-23-O-β-d-glucopyranosyl-28-O-β-d-(6-O-acetyl)-glucopyranosyl hederagenin) (5). Of those, cauloside A (4) displayed the greatest antifungal efficacy against rice blast, TGM and TLB. Colchiside (5) moderately suppressed the development of TLB, but exhibited little effect against the other diseases. The synergistic effects of the isolated compounds against TLB were also assessed. Synergistic and additive interactions were observed between several of the sterol compounds. This study indicated that the crude extracts of, and bioactive substances from, the roots of D. asper suppress TGM and TLB. In addition, cauloside A (4) and colchiside (5) could be used as antifungal lead compounds., (Copyright © 2016 Elsevier B.V. All rights reserved.)