The fungi of the genus, Macrolepiota, are grouped under the family Agaricaceae (division Basidiomycota) and comprise B20 species. Various biological activities of the genus, Macrolepiota, have been reported, including anti-microbial, antioxidant and enzyme (trypsin, monophenolase) activities.1–5 However, few species have been studied with regard to their secondary metabolites. Only several free amino acids, fatty acids and sterols have been reported from Macrolepiota excoriata, Macrolepiota procera and Macrolepiota thacodes. 6 Therefore, as part of a systematic study of Korean mushrooms,7 we investigated the constituents of the fruiting bodies of the mushroom Macrolepiota neomastoidea, widely distributed throughout Korea and other East Asian countries. This is a poisonous mushroom known to cause severe gastrointestinal symptoms, including intestinal irritation, vomiting and profuse diarrhea.8 Thus far, little work has been done on the chemical constituents of M. neomastoidea, except for the isolation of two compounds, lepiotins A and B.9 Recently, we reported the isolation of lepiotin C and (R)-5-hydroxypyrrolidin-2-one, as well as lepiotins A and B.10 As part of a continuing study, we have further isolated a new indole alkaloid named macrolepiotin (1), together with four known ergosterols, (22E,24R)-5a,8a-epidioxyergosta-6,9,22-triene-3b-ol (2),11 (22E,24R)-5a,8a-epidioxyergosta-6,22-dien-3b-ol (3),12 (24S)-ergost-7-en-3b-ol (4)13 and (22E,24R)-5a,6a-epoxyergosta-9(14),22-diene-3b,7a-diol (5).14 In this study, we describe the isolation and structural elucidation of 1 and the cytotoxic activities of compounds 1–5.