Ustusorane D and penicisochromans B-D are natural isochromans isolated from Aspergillus ustus 094102 and Penicillium sp. PSU-F40, respectively. Herein, we report the syntheses of (-)-ustusorane D and (+)-penicisochroman B and the structures of penicisochromans C and D. The relative configuration of natural ustusorane D and the absolute configuration of natural penicisochroman B were determined. Two plausible structures for penicisochroman C were evaluated through synthesis, but their ¹H and ¹³C NMR data were not in agreement with those of the natural product. The structural revision and the determination of the absolute configuration of natural penicisochroman D were achieved. Structure-activity relationship studies of the synthetic compounds as well as a series of related isochromans indicated that the enone of the furanone moiety was essential for the cytotoxicity of these compounds toward HCT116 human colon cancer cells. Pseudodeflectusin, the related natural isochroman, suppressed cell growth and induced apoptosis in HCT116 cells.