Three new hypothemycin analogues were isolated from the fungal strains Hypomyces subiculosus DSM 11931 and DSM 11932. The structures of these compounds were elucidated by spectroscopic methods, chemical conversion, and X-ray crystallographic analysis. One of the analogues, 4-O-demethylhypothemycin, exhibited potent and selective cytotoxic activity against cell lines with a BRAF mutation.