Ethanol-based fuels are becoming more heavily used, increasing the likelihood of ethanol-based fuel spills during transportation and storage. Although ethanol is well-known to be readily biodegradable, very little is known about the effects that such a spill might have on an indigenous microbial community. Of particular concern is that ethanol contamination could stimulate the growth of organisms that can generate regulated compounds and/or produce explosive quantities of methane gas. A column-based study was performed to elucidate the potential impacts of ethanol-based fuel (E85) on the indigenous microbial community during a simulated fuel spill. A continuous dilute supply of E85 resulted in profound shifts in both the bacterial and archaeal communities. The shift was accompanied by the production of high concentrations of volatile fatty acids and butanol, a compound that is regulated in groundwater by some states. Results also indicated that a continuous feed of dilute E85 generated explosive levels of methane within one month of column operation. Quantitative PCR data showed a statistically significant increase in methanogenic populations when compared to a control column. The elevated population numbers correlated to areas of the column receiving a sustained carbon load. Toxicity data indicated that microbial growth was completely inhibited (as evidenced by absence of ethanol breakdown products) at ethanol levels above 6% (v/v). These data suggest that ethanol from ethanol-based fuel can be readily degraded, but can also produce metabolic products that are regulated as well as explosive levels of methane. The core of an E85 spill may serve as a long-term source of contamination as it cannot be degraded until significant dilution has occurred.