Biosurfactants produced by particular bacteria can be applied in petroleum industries to enhance petroleum recovery by lowering the interfacial tension (IFT) between water and oil layers in the reservoir. Pseudomonas aeruginosa was used in the present study to biotransform the pretreatment biodiesel waste into biosurfactant. Biodiesel waste originally contains a number of complex compounds that need to be fragmented into simple ones before used as the substrate for the biotransformation. Pretreatment of the biodiesel waste by ozonation has been chosen in this study due to its simple and fast process. Based on the biosurfactant production yield, the optimum time for the ozonation pretreatment was about 30 minutes. Biosurfactant produced from biotransformation of the pretreatment biodiesel waste by P. aeruginosa was able to reduce IFT of water-oil layer up to 99.09% and the surface tension of pure water up to 27.71 %. These results are close to the biosurfactant criteria for the petroleum recovery.