A pilot-scale aquifer system was used to evaluate groundwater quality impacts from a continuous release of an aqueous 10% v:v ethanol solution mixed with benzene and toluene (50 mg/L each). The geochemical footprints (methane, volatile fatty acids (VFAs), pH, oxidation reduction potential (ORP), dissolved oxygen (DO) and temperature) were monitored over 11 months. The accumulation ofVFAs (particularly butyric acid) during the summer exceeded the secondary maximum contaminant level value for odor, which represents a previously unreported aesthetic impact. Temperature variations (3.9 to 30.0 °C) significantly affected microbial activities, and a strong correlation was observed between groundwater temperature and CH4/VFAs generation (p < 0.05). Quantitative real-time PCR (qPCR) analysis showed that ethanol blend increased the microbial population and enriched the fermentative groups for acetogenesis and methanogenesis.