Nutritional requirements can contribute considerably to the production cost and the bioprocess economics. Media optimisation using response surface methodology is one of the used methods to ameliorate the bioprocess economics. In the present study, biosurfactant production by Bacillus subtilis SPB1 was effectively enhanced by response surface methodology. A Plackett-Burman-based statistical screening procedure was adopted to determine the most important factor affecting lipopeptide production. Eleven variables are screened and results show that glucose, K(2)HPO(4), and urea concentrations influence the most biosurfactant production. A Central Composite Design was conducted to optimize the three selected factors. Statistical analyses of the data of model fitting were done by using NemrodW. Results show a maximum predicted biosurfactant concentration of 2.93 (±0.32) g/L when using 15 g/L glucose, 6 g/L urea, and 1 g/L K(2)HPO(4). The predicted value is approximately 1.65 much higher than the original production determined by the conventional one-factor-at-a-time optimization method.