From Penicillium janczewskii, obtained from a marine sample, two new diastereomeric quinolinones, 3S*,4R*-dihydroxy-4-(4′-methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (1) and 3R*,4R*-dihydroxy-4-(4′ methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (2), were identified, along with two known alkaloids, peniprequinolone (3) and 3-methoxy-4-hydroxy-4-(4′-methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone (4). Penicillium janczewskii Zalessky (strain H-TW5/869) was selected from our screening program for further studies, because of a rich metabolite pattern as detected by thin-layer chromatography on silica gel with various staining reagents, and cytotoxic activity of its methanolic extracts. The extracts from mycelia and culture filtrate showed significant cytotoxicity against CHO-K1 cells. In addition to two known fungal metabolites (3 and 4), our studies yielded new diastereomeric dihydroquinoline alkaloids 1 and 2. We here report the isolation and structure elucidation of 1 and 2, as well as the biological activity of these compounds. Cytotoxicity testing on eight tumor cell lines revealed a moderate specificity of 2 on SKOV-3 cells.