Abstract: In the present study Pseudomonas aeruginosa PB3A strain isolated from oil contaminated soil was exploited for the biosurfactant production. Effect of growth parameters for the biosurfactant production were studied which revealed their optimum production occurs at pH 7, with a temperature of 37°C and optimum incubation time of 48 h. Further the characterization studied of partially purified biosurfactants by TLC and FTIR confirms the presence of biosurfactant production. Screening of antibacterial and anticancer studies also reveals that, the extracted biosurfactant effectively controlled the test pathogens and also suppress the proliferation of HeLa cells showing an IC50 value of 125 μg mL–1. The present study concludes the biosurfactant produced by Pseudomonas aeruginosa PB3A shows significant cytotoxicity activity which is of eco-friendly in nature.