In order to provide more natural antifouling compounds, marine bacterium Pseudomonas putida isolated from the sponge Haliclona sp. was explored to test its anti-diatom compounds. METHODS AND RESULTS: The strain was identified by colonial morphology, scanning electron microscope (SEM) and 16S rDNA sequence analysis. The separation procedure was guided by bioactive (Anti-diatom) and chemical (TLC, DAD-HPLCand 1H NMR) analysis, and their structures were elucidated by spectrographic techniques. The anti-diatom activity of all purified compounds was assayed. Strain 272 isolated from the sponge Haliclona sp. was identified as Pseudomonas putida. Six diketopiperazine compounds were isolated from the culture of this strain and their structures were determined as cyclo(Leu-Pro) (1), cyclo (Leu-Ala) (2), cyclo(Phe-Ala) (3), Cyclo(Tyr-Val) (4), cyclo(Ala-Tyr) (5), cyclo(Ala-Trp) (6); Compounds 3 and 6 displayed significant anti-diatom activity with the inhibitory rate of 50% and 85% at the concentration of 50 microg/mL, respectively. CONCLUSIONS: The anti-diatom compounds isolated from marine bacterium Pseudomonas putida were cyclo (Phe-Ala) and cyclo (Ala-Trp).