Bacteria associated with surfaces have been frequently cited as a potential source for the isolation of bioactive metabolites. In this study, bacteria associated with marine gastropod, Babylonia sp. were isolated and screened for antibacterial activity against biofilm-forming bacteria. The antibiofilm and antifouling effect of the selected surface- associated bacterial strains were examined under in vitro and in vivo conditions. Results showed that the extracellular polymeric substances (EPS) of the bacterial strain CML associated with gastropod species considerably reduced the adhesion of biofilm-forming bacteria on glass coupons. Besides, the antifouling coat prepared by incorporating of this EPS into polyurethane varnish prevented the settlement of biofoulers on test substratum submerged in marine waters. The functional groups present in the EPS were analyzed using FT-IR. The bacterium responsible for the production of the bioactive EPS was identified as Bacillus subtilis subsp. by 16S rRNA gene sequencing. More detailed characterization of the identified bioactive EPS could lead to the isolation of a novel natural antifouling product.