In the course of searching for new anticancer compounds from marine microorganisms, a new natural product iso-α-cyclopiazonic acid (1), together with its isomer α-cyclopiazonic acid (2), three mycotoxins (aflatoxin B1 (3), aflatoxin Q1 (4), O-methylsterigmatocystin (5)), and two diketopiperazine alkaloids (ditryptophenaline (6), 3-[(1H-indol-3-yl)methyl]-6-benzylpiperazine-2,5-dione (7)) were isolated from the marine-derived fungus Aspergillus flavus strain c-f-3, which was obtained from marine algae collected in Putian Pinghai, China. Compound 1 was first synthesized by Holzapfel in 1968 and is reported here as a natural product for the first time. Their structures were elucidated using spectroscopic techniques including ESI-MS, 1H/13C NMR, and CD. Comparative analysis revealed that 1 and 2 have identical planar structures but differ in the stereochemistry of C-5: 2 has an S configuration, while 1 has an R configuration. The cytotoxic activities of 1 and 2 were assessed against HL-60 and MOLT-4 cell lines (via MTT assay) and A-549 and BEL-7402 cell lines (via SRB assay). Compound 1 exhibited IC50 values of 90.0, 68.6, 42.2, and >100 μM against the four cell lines, respectively. In contrast, compound 2 showed more potent cytotoxicity with IC50 values of 2.4, 12.3, 21.5, and 17.5 μM, respectively, and was significantly more active than 1 across all tested cell lines. These results indicate that the S configuration at the C-5 position in cyclopiazonic acid is a key determinant of its biological activity. This paper describes the isolation and structural characterization of 1, as well as the cytotoxic activities and structure-activity relationships of 1 and 2.