This work aims to investigate biosurfactant production by a bacterium capable of utilizing spent engine oil. The effects of pH and temperature on the growth of this bacterium on spent engine oil were determined. The bacterium was isolated from petroleum-contaminated soil. Based on 16S rRNA gene sequence, it was identified as Enterobacter cloacae with 99% identity. Growth of Enterobacter cloacae B14 on mineral medium containing 1% (v/v) spent engine oil was optimum when incubated at 30oC. The bacterium showed the ability to grow under a wide range of pH medium, whereas the highest specific growth rate (0.29 hr^-1) was obtained when grown under pH 7. Biosurfactant production by E. cloacae B14 was observed when grown in mineral medium containing 1% (w/v) glucose. Cell-free supernatant showed 42.2% emulsification activity (E24) against spent engine oil. Such activity was higher than some previous works and was obtained from a lower amount of biomass. Positive results from oil displacement, drop collapse and CTAB-methylene blue agar tests strongly indicated that strain B14 is an effective biosurfactant producer. Therefore, Enterobacter cloacae B14 has the potential for applications in bioremediation of oil-contaminated sites due to its biosurfactant production and growth on spent engine oil.