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. © 2007 The Authors.