A marine isolate of Micrococcus MCCB 104 has been identified as an aquaculture probiotic antagonistic to Vibrio. In the present study different carbon and nitrogen sources and growth factors in a mineral base medium were optimized for enhanced biomass production and antagonistic activity against the target pathogen, Vibrio harveyi, following response surface methodology (RSM). Accordingly the minimum and maximum limits of the selected variables were determined and a set of fifty experiments programmed employing central composite design (CCD) of RSM for the final optimization. The response surface plots of biomass showed similar pattern with that of antagonistic activity, which indicated a strong correlation between the biomass and antagonism. The optimum concentration of the carbon sources, nitrogen sources, and growth factors for both biomass and antagonistic activity were glucose (17.4 g/L), lactose (17 g/L), sodium chloride (16.9 g/L), ammonium chloride (3.3 g/L), and mineral salts solution (18.3 mL/L).