Groundwater at many industrial polluted sites is usually contaminated by complex contaminants including petroleum hydrocarbons and heavy metals. In this study, the effectiveness of sulfate-reducing mechanisms on toluene and copper contaminated groundwater cleanup was evaluated in microcosm experiments. Sulfate was supplied into microcosms containing toluene (17.5 mg/L) and copper (12 mg/L) contaminated groundwater to activate the sulfate reducing process. The inocula used in the microcosms contained petroleum-hydrocarbon contaminated soils and sludge collected from an anaerobic basin of a wastewater treatment facility. Approximately 99% of toluene and copper could be removed during the 40-day operational period with the decay rates of 0.19 and 0.12 1/d, respectively. Under sulfate-reducing mechanisms, toluene could be biodegraded and a consumption of 0.105 g/L of sulfate was observed. Copper removal (dropped to below 0.1 mg/L) was due to the bioprecipitation mechanisms, which could be confirmed by the occurrence of sulfate reduction mechanisms (sulfide increased from 8.6 to 686 μg/L) and copper sulfide formation. Increased hydrogen sulfide concentrations also resulted in the inhibition of microbial growth. Dominant bacterial species and microbial communities were characterized by molecular biological technologies. A total of 12 dominant sulfate-reducing bacteria and petroleum-hydrocarbon degraders were detected. Results show that simultaneous toluene and copper removal could be achieved via the sulfate reduction and heavy metal bioprecipitation mechanisms. Sulfate reduction became the predominant biodegradation mechanism after sulfate supplement under anaerobic conditions. Results from this study can be applied to develop a cost-effective bioremedial system to cleanup heavy-metal and petroleum-hydrocarbon polluted groundwater.