A new peptaibol, RK-026A (1) was isolated from a fungus, Trichoderma sp. RK10-F026, along with atroviridin B (2), alamethicin II (3), and polysporin B (4) as a cytotoxic compound, which was selected by principal component analysis of the MS data from 5 different culture conditions. The structure of 1 was determined as a new atroviridin B derivative containing Glu at the 18th residue instead of Gln by NMR and HR-MS analyses including the investigation of detailed MS/MS fragmentations. 1 showed cytotoxicity toward K562 leukemia cells at an IC50 value of 4.1 µm.