Aim: To develop a new medium for enhanced production of biomass of an aquaculture probiotic Pseudomonas MCCB 103 and its antagonistic phenazine compound, pyocyanin. Methods and Results: Carbon and nitrogen sources and growth factors, such as amino acids and vitamins, were screened initially in a mineral medium for the biomass and antagonistic compound of Pseudomonas MCCB 103. The selected ingredients were further optimized using a full-factorial central composite design of the response surface methodology. The medium optimized as per the model for biomass contained mannitol (20 g l(-1)), glycerol (20 g l(-1)), sodium chloride (5 g l(-1)), urea (3.3 g l(-1)) and mineral salts solution (20 ml l(-1)), and the one optimized for the antagonistic compound contained mannitol (2 g l(-1)), glycerol (20 g l(-1)), sodium chloride (5.1 g l(-1)), urea (3.6 g l(-1)) and mineral salts solution (20 ml l(-1)). Subsequently, the model was validated experimentally with a biomass increase by 19% and fivefold increase of the antagonistic compound. Conclusion: Significant increase in the biomass and antagonistic compound production could be obtained in the new media. Significance and Impact of the Study: Media formulation and optimization are the primary steps involved in bioprocess technology, an attempt not made so far in the production of aquaculture probiotics.