Ergosterol is an essential component for fungi, including entomopathogenic fungi like Cordyceps. Cordyceps has been used as a traditional medicine in Japan and China and possesses various unique ergostane-type sterols, those exhibit bioactivities. In this manuscript, we reported the isolation of 2 new ergostane-type sterols, 1 and 2 along with ten sterols (3-12) from 7 strains of Cordyceps related fungal strains, Cordyceps takaomontana NBRC 101754, Metarhizium owariense NBRC 33258, Polycephalomyces formosus NBRC 109994, Cordyceps tuberculata NBRC 106948, Cordyceps tenuipes NBRC 108997, Cordyceps sp. NBRC 106954, and Tolypocladium paradoxum NBRC 106958 collected fruiting bodies of Ophiocordyceps heteropoda. In addition, the antitrypanosomal activity and antimicrobial activity of isolates were tested to find 6 showed the antitrypanosomal activity, and the minimum inhibitory concentration (MIC) value was confirmed as 1.41 µg/mL. In the antimicrobial assay, the MIC value of 8 against methicillin-resistant Staphylococcus aureus was determined to be 3.1 µg/mL.