Wakayin (3) is the first reported example of a pyrroloiminoquinone alkaloid to be isolated from an ascidian. Ascidians have been the recent source of many iminoquinone-bearing polycyclic aromatic alkaloids. These structurally related compounds typically exhibit a wide range of biological activities, including murine cell-line cytotoxicity, topoisomerase II, and microbe/fungus inhibition. The related pyrroloiminoquinone alkaloids, isobatzellines (e.g., isobatzelline C (1)), and discorhabdins/prianosins (e.g., discorhabdin C (2)) are, however, known only from phylum Porifera. In continuation of our search for biologically active secondary metabolites from ascidians, we now report the structure of a new pyrroloiminoquinone derivative, wakayin (3), isolated from the ascidian Clavelina sp. Wakayin exhibited in vitro cytotoxicity against the human colon tumor cell line (HCT116 IC₅₀ 0.5 μg/mL). Inhibition of topoisomerase II enzyme (250 μM) and the observation of a 3-fold differential toxicity toward the CHO cell line EM9 (sensitive to DNA-damaging genotoxic agents) versus BR16 (resistant to BCNU) provided preliminary evidence that wakayin exhibits its cytotoxicity by interfering with or damaging DNA. Antimicrobial activity against Bacillus subtilis (MIC = 0.3 μg/mL) was also observed.