Abstract: Marine fouling organisms pose severe economic problems. However, the traditional metal-based antifoulants we used usually pollute marine environment. Thus, environmentally friendly alternatives are urgently needed. Natural antifouling compounds are important sources for developing environmentally friendly antifoulants. In this study, we investigated the potential use of the fermentation extract (FE) from the bacterium Vibrio fluvialis MS-30 as natural antifoulants by assessing its antifouling activity using the settlement assay with the cyprids of the barnacle Balanus albicostatus and the field assay. Our results showed that the fermentation extract exhibited antifouling activity against settlement of B. albicostatus at 25 μg/mL. The coatings containing FE (10% and 20%) exhibited antifouling activity against microfouling and macrofouling in the field. Furthermore, the optimized fermentation conditions for obtaining the high extraction rate of FE from the strain MS-30 were found to be 3% for inoculum volume, 28 for salinity, 72 h for fermentation time, and 26 ℃ for fermentation temperature. The extraction rate and antifouling activity of FE extracted with ethyl acetate were significantly higher than those extracted with n-butanol and petroleum ether. This study provides insights for the potential use of FE from the MS-30 strain in the field of marine antifouling technology.