Two potential biosurfactant producing microorganisms - Acinetobacter Junii and Pseudomonas aeruginosa were Isolated from oil contaminated soils and air. The aim of this work was to evaluate the comparative potential of these two microorganisms in utilizing different vegetable and mineral oils as substrate for production of biosurfactant. The effect of pH and temperature on biosurfactant production by these two microorganisms was also evaluated. High molecular weight, high boiling mineral oils e.g. diesel resulted in better surface activity with Pseudomonas aeruginosa and produced rhamnolipid type of biosurfactant. The trend was reverse for Acinetobacter junii which produced proteoglycan type biosurfactant of comparatively less activity using petrol (low molecular weight and low boiling mineral oil fraction) as substrate. In case of vegetable oils, unsaturated C16 and C18 containing substrates were more effective for biosurfactant production especially for Pseudomonas aeruginosa. Optimum pH for Acinetobacter junii was 7.0 where as optimum growth of Pseudomonas aeruginosa was observed at pH 7.5. Optimum temperature for growth of both microorganisms was 30 °C. This study has thus screened and Identified two microorganisms viz. Pseudomonas aeruginosa and Acinetobacter junii for biosurfactant production using different vegetable and mineral oils as substrate. The biosurfactant produced by Pseudomonas aeruginosa had better surface activity considering both surface tension and emulsiflcatlon Index. © 2014 Scientific Publishers.All Rights Reserved.