A selected Pseudomonas aeruginosa strain was grown on the basal medium and inspected for rhamnolipid biosurfactant production. In order to increase the rhamnolipid production, limitations of both magnesium and iron elements in the basal medium were tested. The Fe concentrations of 0.05 g/L and above inhibited the increase in both cell growth and rhamnolipid production. Mg concentration had a negligible effect on rhamnolipid levels than Fe. While Carbon/Mg ratios were between 400-2000, biosurfactant concentration increased and when Carbon/Fe ratio was above 5000, biosurfactant production increased until the ratio reached about to 105. In addition to the nutrients, effects of pH and temperature were also investigated. Culture broths were incubated at between 20-40oC. Maximum rhamnolipid production and cell growth were observed at 34.5oC. Optimum pH values for rhamnolipid production were found between 6.5-7.0