A small library of soluble 2,5-diketopiperazine derivatives were designed, synthesized and evaluated as antifouling agents against two species of marine organisms: Balanus amphitrite and Bugula neritina by larval attachment assay. The results showed that different derivatives had different antifouling activities. Among these compounds, 3a (EC50 = 2.5 mu g mL(-1)), 3b (EC50 = 3.2 mg mL(-1)) and 3d (EC50 = 1.6 mg mL(-1)) had strong activities, and 2d (EC50 = 20.5 mg mL(-1)), 3l (EC50 = 25.0 mg mL(-1)) and 3m (EC50 = 18.5 mg mL(-1)) had moderate activities against Balanus amphitrite, but other compounds had no activities. Differently, compounds 3d (EC50 = 3.1 mg mL(-1)) and 3i (EC50 = 2.3 mg mL(-1)) exhibited strong activities, and 3a (EC50 = 11.3 mg mL(-1)) and 3c (EC50 = 17.2 mg mL(-1)) showed moderate activities against Bugula neritina, whereas other compounds had no activities. All of the active compounds had stronger antifouling activities than those of the natural product cyclo-(L-Phe-L-Pro). Compound 3d could be a new template molecule for further development as an antifouling agent because of its strong inhibitory activities against both species of fouling organisms with low or no toxicity to the environment.