In our ongoing search for structurally interesting and biologically active metabolites from Korean wild mushrooms, bioassay-guided fractionation and a chemical investigation of the MeOH extracts of the fruiting bodies of the mushroom Naematoloma fasciculare resulted in the isolation of three ergosterol derivatives, (22E,24R)-ergosta-7,22-diene-$3{eta}$,$5{alpha}$,$6{eta}$,$9{alpha}$-tetrol (1), (22E,24R)-$5{alpha}$,$8{alpha}$-epidioxyergosta-6,22-diene-$3{eta}$-ol 3-O-${eta}$-$small{D}$-glucopyranoside (2), and (22E,24R)-$5{alpha}$,$8{alpha}$-epidioxyergosta-6,9,22-triene-$3{eta}$-ol 3-O-${eta}$-$small{D}$-glucopyranoside (3). The structures of 1 - 3 were determined by comparison of their spectroscopic and physical data with reported values. The isolated steroid derivatives 1 and 3 were reported for the first time from this mushroom. Compounds 1 - 3 were tested for their cytotoxic activities against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15).