Numerous bioactive nucleoside analogs have been isolated from terrestrial and marine sources and some of them are expected to be clinically useful compounds. During our continuing search for new bioactive substances from Okinawan marine organisms, we recently examined extracts of the Okinawan tunicate Aplidium multiplicatum Sluiter and isolated a novel nucleoside derivative, shimofuridin A (1), containing an acylfucopyranoside moiety and possessing cytotoxic and antimicrobial activities. This paper describes the isolation and structure elucidation of 1. Shimofuridin A (1) exhibited cytotoxicity against murine lymphoma L1210 cells with an IC50 value of 9.5 μg/mL in vitro and antimicrobial activity against fungus Trichophyton mentagrophytes (MIC value, 133 μg/mL) and Gram-positive bacterium Sarcina lutea (MIC, 66 μg/mL). Compound 1 also showed inhibitory activity against protein kinase C (IC50 20.0 μg/mL).