||In the marine environment, all submerged surfaces are constantly exposed to and colonized by microfoulers (bacteria, diatoms, and protozoa) and macrofoulers (invertebrates larvae, and macroalgal spores). To control biofouling in marine industry, new and environmentally friendly biofouling agents are under urgent demand due to recent ban on organotin products. Natural products from marine bacteria are a new frontier for biotechnology development. Deep-sea microorganisms are a new source of bioactive compounds. In this study, I screened about 200 bacterial strains isolated from deep-sea sediments for their antifouling activity. After identifying the bioactive bacterial strains, I cultured two different isolates at large-scale and purified bioactive compounds using bioassay-guided fractionation. A branched-chain fatty acid as well as a group of butenolides with strong antifouling activity were identified and chemically characterized. To analyze structure-activity-relationships of these antifouling compounds, I also acquired the analogues of these compounds with an ultimate goal of identifying functional group of those compounds and the chemical structure with the most desirable antifouling activity. Since the culture conditions often have great impact on the production of bioactive compounds in bacterial suspension culture, to achieve a high yield of those compounds, I optimized the components in bacterial culture media and the physical conditions under which the bacteria may achieve high yield of a target butenolide. Furthermore, to better understand how the bioactive compounds inhibit larval settlement process, I investigated the changes in genes and proteins differentially expressed in settling larvae that were exposed to the bioactive compounds in comparison to those untreated with the antifouling compounds, suggesting the possible roles of these genes and proteins in regulating larval settlement and metamorphosis. My results clearly demonstrated that 1) deep-sea microbes are potent but underexplored source of bioactive compounds, 2) bioactive compounds identified by me have a great market potential as non-toxic antifouling agents, 3) mode-of-action of bioactive compounds against target organisms are new frontier research in biofouling/antifouling research in near future, and 4) multi-disciplinary approach is a MUST strategies in antifouling technology development.