In the course of our mycochemical studies the extract of Clitocybe nebularis was investigated with the aim to identify its bioactive secondary metabolites. Multistep chromatographic purification of the MeOH extract of C. nebularis resulted in the isolation of two steroids and an organic acid from the CHCl3 and ethyl acetate soluble fractions. The structures of the compounds were determined by NMR and MS spectroscopy as 5α-ergosta-7,22-diene-3β,5,6β-triol (cerevisterol) (1), (22E,24S)-5α-ergosta-7,22-diene-3β,5,6β,9α-tetraol (2), and indole-3-carboxylic acid (3). The antimicrobial activity of the compounds was analyzed by agar disc diffusion method against human pathogen strains of Streptococcus agalactiae, Staphylococcus epidermidis, Moraxella catarrhalis, Haemophilus influenzae, and Proteus mirabilis. The susceptibility assay revealed that compounds 2 and 3 have weak antimicrobial activity against M. catarrhalis. The current study represents the first isolation of compounds 1-3 from C. nebularis.