A polycyclic tetrahydroxanthone, actinomadurone, was isolated from the actinomycete Actinomadura sp. BCC 35430. Its structure was elucidated by extensive spectroscopic analyses. Actinomadurone showed antifungal activities against Curvularia lunata, Alternaria brassicicola, Colletotrichum capsici, and Colletotrichum gloeosporioides; and displayed cytotoxic activity against Vero cell lines.