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The primary motive of this research was to check for the presence of microbial species which has the ability to produce biosurfactant. The soil samples were collected from petrol bunk which was contaminated with petroleum oil and enrichment technique was carried out with minimal salt medium (MSM) for isolation of microbial strain. Serial dilutions of the enriched culture were performed. Two bacterial isolates and one fungal isolate showed activity for biosurfactant production. The methods employed for screening were oil displacement, oil drop collapse, blood hemolysis, blue agar plate method and confirmatory tests like phenol-sulphuric acid method. Both, bacterial isolates (MB1 and MB2) and fungal isolate (JAD1) showed emulsification activity of 41.66 %, 64.70 % and 90.90 % respectively. Biosurfactant was extracted and screened for antimicrobial activity against Gram positive and Gram-negative clinical pathogens. These biosurfactant producing isolates can be used to enhance in situ bioremediation of soils contaminated with polycyclic aromatic hydrocarbons (PAHs).