Surfactants are amphipathic molecules which reduce surface tension, are widely used in pharmaceutical, cosmetic and food industries. In the present study, Streptomyces coelicoflavus, the biosurfactant producing actinomycetes, was isolated frogm soil contaminated with petroleum oil near the naval dockyard, Visakhapatnam. This isolate was then grown in Kim’s medium for extracellular biosurfactant production, and was evaluated by measuring emulsification index, surface tension, lipase production and para film-M method. Further quantification of Rhamnolipid biosurfactant was done by orcinol assay. The Plackett-Burman design was adopted to evaluate the amount of biosurfactant produced. Among the seven variables of Kim’s medium, three variables, namely, concentration of olive oil, NaNO3 and level of inoculums, were identified to cause a significant effect on the biosurfactant production. When the strain Streptomyces coelicoflavus was cultivated in the optimized medium, the biosurfactant production was increased to 475.24 µg/ml compared to un-optimized medium which was 458.78 µg/ml.