A microbial survey of hydrocarbon-impacted groundwater and vadose zone at a Midwestern refinery employed molecular biological tools to elucidate the microbial processes involved in bioremediation occurring in the subsurface. qPCR analysis of bio-traps incubated in groundwater indicated that a large and diverse microbial community was present throughout the site and suggested that mechanisms of benzene, toluene, ethylbenzene, and xylene (BTEX) biodegradation included aerobic oxidation, sulfate reduction, methanogenesis, and possibly Fe+3 reduction. To assess the role of vadose zone microorganisms in hydrocarbon attenuation, RNA was extracted from soil core samples, and reverse transcriptase-qPCR (RT-qPCR) analysis indicated that microbial activity in the vadose zone generally increased with depth, likely supported by hydrocarbons and methane volatilizing from the groundwater. Stable isotope probing (SIP) with 13C6-benzene provided direct evidence of benzene biodegradation in six of the eight wells studied. The highest levels of 13C were detected in dissolved inorganic carbon (DIC) extracted from the two monitoring wells closest to the river. The influx of nutrients and oxygen coming from the river may help to maintain a robust population of hydrocarbon degraders in these wells. While qPCR analysis indicated that microorganisms with the genetic potential for hydrocarbon biodegradation were ubiquitous at the site, RT-qPCR and SIP results were used to refine the site conceptual model by identifying areas where that genetic potential was actively being expressed and locations where biodegradation was lagging.