Three antifouling active compounds of L-pyroglutamic acid (PGA), triethyl citrate (TEC) and di-n-octylphthalate (DNOP) were isolated from the brown seaweed Ishige okamurae. Approximately 2.8 mg PGA, 1.7 mg TEC, and 2.0 mg DNOP were isolated from 600 g of I. okamurae powder. The concentrations of PGA, TEC, and DNOP required to cause foot repulsion in 50% of mussels (RD50) were 9, 26, and 0.08 mM, respectively. The PGA, TEC, and DNOP concentrations required to inhibit 50% attachment of algal spores (ID50) were 24, 50 and 0.1 mM, respectively. These compounds showed stable antifouling activities against mussel and algal spore attachment.