Nutritional requirements for maximal production of biosurfactant by an oil field bacterium Pseudomonas putida were determined. The optimal concentrations of nitrogen, phosphate, sulfur, magnesium, iron, potassium, sodium, calcium, and trace elements for maximal production of biosurfactants were ascertained, and a new “Pruthi and Cameotra” salt medium was formulated. Data show that maximal biomass (2.4 g L⁻¹) and biosurfactant production (6.28 g L⁻¹) takes place after 72 h of growth on 2% hexadecane. The biosurfactant was produced optimally over pH and temperature ranges of 6.4–7.2 and 30–40°C, respectively. That the highest biosurfactant yield was obtained during late log phase of growth indicates that the biosurfactant is a secondary metabolite.