Incomplete combustion of fossil fuels and other anthropogenic activities result in contamination of surface water by petroleum hydrocarbons. These pollutants can have severe effects on aquatic life and human health. In petroleum bioremediation, oil degrading microorganisms are utilized to remove petroleum hydrocarbons from polluted water. However, monitoring and identifying microorganisms capable of degrading petroleum hydrocarbons is very challenging. In the current study, bacteria isolated from a river alongside a petroleum refinery were assessed for their petroleum hydrocarbon degradation abilities. Primary selection was performed through traditional enrichment culture method, emulsifying ability measurement, and gravimetric analysis of hydrocarbon degradation. Screening of two novel hydrocarbon catabolism genes encoding key enzymes in hydrocarbon degradation, alkB and C23O, was performed by using polymerase chain reaction. The bacterial isolates, degrading hydrocarbons with high emulsification abilities and harboring the catabolic genes, were identified as species of Acinetobacter by 16S rRNA sequence analysis. Monitoring the temporal distribution of Acinetobacter in river water was also done by fluorescence in situ hybridization using DNA probe ACA652. The results revealed that species of Acinetobacter successfully populate the polluted surface water and have high potential for petroleum bioremediation.