Penicimutanin C, a new alkaloidal compound, was isolated from the neomycin-resistant mutant strain 3-f-31 of the marine-derived fungus, Penicillium purpurogenum G59, together with four known compounds. The structure of penicimutanin C, including the absolute configuration, was determined by spectroscopic and chemical methods. The absolute configuration of penicimutanin A was also re-confirmed by Marfey's and chiral HPLC analyses of the hydrolyzed products. Penicimutanins C and A inhibited the proliferation of five human cancer cell lines to some extent. Penicimutanin C is the third dimer of diketopiperazine and penicimutanolone, which are only produced by mutants of P. purpurogenum G59 isolated to date, and it showed cytotoxic activity against human cancer cell lines. The neomycin-resistant screening strategy has been previously successfully used to discover new compounds by activating silent metabolites in fungi, and the present results provide an additional example of the effectiveness of this method. © 2020 Wiley-VHCA AG, Zurich, Switzerland