Central composite rotatable design (CCRD) of experiments was used to obtain data for Lipopeptide and Biomass concentrations from fermentation medium containing the following five components: glucose, monosodium glutamate, yeast extract, MgSO4⋅7H2O, and K2HPO4. Data was used to develop a second order regression response surface model (RSM) which was coupled with ant colony optimization (ACO) to optimize the media compositions so as to enhance the productivity of lipopeptide. The optimized media by ACO was found to yield 1.501 g/L of lipopeptide concentration which was much higher compared to 1.387 g/L predicted by Nelder–Mead optimization (NMO). The optimum from ACO was validated experimentally. RSM-based ACO is thus shown to be an effective tool for medium optimization of biosurfactant production. © 2016, Indian Academy of Sciences.