The application of bark-compost to farm land has been reported to suppress soil-borne diseases, including Fusarium wilts. The "bacterial type" microflora of the compost, that includes many antagonists, has been considered to play the major role in suppression of soil-borne plant pathogens. To elucidate the suppressive effect of hemlock bark-compost on soil-borne pathogenic fungi, one of the antifungal agents in bark-compost was isolated by preparative HPLC. From 2D-nuclear magnetic resonance spectra and high resolution mass spectrometry, the antifungal agent was determined to be 24R-ethylcholest-5-en-3β-ol (silosterol). Sitosterol is a normal secondary plant metabolite found throughout the plant kingdom. Furthermore, using reverse-phase HPLC, we found considerable amounts of cholesterol, campesterol and stigmasterol in acetone extracts of the bark-compost. These typical sterols had strong in vitro antifungal activity against Fusarium oxysporum f. sp. cucumerinum. We investigated the antimicrobial spectrum of cholesterol. It was active against Cochliobolus miyabeanus, Gibberella zeae, Glomerella cingulata, Alternaria alternata and Rhizoctonia solani, fungi of interest to plant and human pathologists. It was inactive against yeasts and procaryotic organisms, based on a disk-diffusion technique. We propose that there might be a parallel between this selective antimicrobial activity of the sterol and the formation of a "bacterial type" microflora of the compost. These findings suggest that the typical sterols, such as cholesterol, may function as defensive substances against pathogenic fungi at the external surface of higher eucaryotes, including the cuticle of higher plants and in kelatinized squames of the mammalian epidermis. © 1990.