We report on the structure elucidation of arnamial, a new Delta(2,4)-protoilludane everninate ester from the fungus Armillaria mellea, and on the apoptotic activity of arnamial as well as the cytotoxic activity of structurally related compounds on selected human cancer cells. Arnamial showed cytotoxicity against Jurkat T cells, MCF-7 breast adenocarcinoma, CCRF-CEM lymphoblastic leukemia, and HCT-116 colorectal carcinoma cells at IC50 = 3.9, 15.4, 8.9, and 10.7 microM, respectively, and the related aryl ester melledonal C showed cytotoxic activity against CCRF-CEM cells (IC50 = 14.75 microM). [1,2-13C2]Acetate feeding supports a polyketide origin of the orsellinic acid moiety of arnamial.