We developed three new assay methods: an antialgal test using the diatoms, Nitzschia closterium and Naviculla sp.; a growth-inhibition assay against invertebrate larvae of Musculista senhousia; and an assessment of the residual amount of tested samples on conventional submerged assay plates used for screening antifouling substances. A combination of these assay procedures with the antimicrobial assay previously reported resulted in establishing a laboratory-maching, more reliable and time-saving assay system for antifouling substances.