Rhamnolipids are interesting microbial surfactants having great industrial importance. However, the main obstacles towards an economic production of rhamnolipids are low productivity and high raw‐material costs. Therefore, this study aimed at optimization of the culture media as well as culture conditions using response surface methodology for maximum rhamnolipid production by Pseudomonas aeruginosa isolate P6, a promising rhamnolipid‐producing isolate. The optimum medium for maximum rhamnolipid production was found to be a mineral salts medium with glycerol 2 % v/v as the carbon source. The optimum cultivation conditions using response surface methodology were found to be an incubation temperature of 30 °C, an agitation rate of 250 rpm, an inoculum size of 5 % v/v and unlike most studies, an initial pH of 7.5. The resulting model predicted data points that corresponded well to the experimental values. Optimization resulted in a threefold increase in rhamnolipid production reaching 7.54 g/L. The data are potentially useful for further industrial exploitation of rhamnolipid production by the studied isolate.