We evaluated the biological activities of five water extracts of tissue of the mottled anemone Urticina crassicornis. Most extracts exhibited broad-spectrum antimicrobial activity as determined by ultrasensitive radial diffusion assay (URDA) against grampositive and -negative bacteria, including a fish pathogen, Aeromonas hydrophila, but no activity against fungi. The activity of the extracts was abolished by tryptic digestion, indicating that protein compounds were responsible for the antimicrobial activity. Furthermore, in a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity assay, only the visceral tissue extract showed activity. However, no extract had hemolytic activity against human red blood cells. Consequently, this study suggests the water-soluble extract of mottled anemone to be a promising source of proteinaceous antimicrobial compounds that can be utilized for development of novel antibiotics.