Metabolites of marine derived fungus Eurotium rubrum MPUC136 differed between cultivation on wheat medium and Czapek-Dox agar medium. Melanin synthesis inhibitory activity of crude extract of culture on wheat medium showed stronger activity than that of crude extract of culture on Czapek-Dox agar medium. A new diketopiperazine compound isoechinulin D (1) and eight reported diketopiperazines (2-9) were isolated from the crude extract of wheat medium. The structure of 1 was established using NMR, MS and IR methods. 2-5 inhibited melanogenesis using B16 melanoma cells (IC50 = 68, 2.4, 83, 9.1 mu M each). Structure-Activity-Relationships of diketopiperazines (1-10) indicated the importance of the prenyl groups at C-2, C-5 and C-7, the vinyl group at C-12 to C-25 and the sp(2) carbons at C-8 and C-9. Isolated compounds (1-9) were not or slightly observed from the extracts of Czapek-Dox agar medium by HPLC analysis, suggesting that different cultivation processes could affect metabolism and enhance bioactivities. (C) 2016 Elsevier Ltd. All rights reserved.