Biosurfactants are amphiphilic compounds produced by microorganisms with almost similar properties as chemically synthesized surfactants. With amphiphilic properties, these compounds are widely applied as bioemulsifier and enhancer in the bioremediation process. This study aimed to screen potential biosurfactant producers from petroleum sludge. Bacteria were isolated from petroleum sludge samples and screened for biosurfactant activity by analysing Emulsification activity (E24) and Surface Tension of the culture supernatant. Selected isolates were subjected to 16S rRNA analysis while the isolate that showed the highest surface tension reduction was further subjected to the growth study. The effect of carbon and nitrogen source towards surface tension reduction was tested. Then, the biosurfactant produced was partially purified and characterized. Out of the 47 isolates, Pseudomonas sp. P15 exhibited among the highest emulsification index (67.41%) and biosurfactant activity by reducing the surface tension measurement as low as 18.92 mN/m. In the bacterial growth study, Pseudomonas sp. P15 exhibited the highest surface tension reduction when 2% of used engine oil (UE2) was used as the carbon source. The addition of nitrogen source, 0.5% of yeast extract did not affect the surface tension reduction but increased the bacteria growth and biosurfactant yield by 100-fold (43.37 g/L). After liquid-liquid extraction, the partially purified biosurfactant showed similar Rf value to the rhamnolipid standard in the thin layer chromatography (TLC) and effectively emulsified the hydrocarbon substrate. The finding indicated that Pseudomonas sp. P15 isolated from petroleum sludge has potential to produce biosurfactant that can be used in wide range of industrial application such as bioremediation of petroleum contaminated sites and enhance the oil recovery in the petroleum industry.