With increased application of co-solvent flushing technologies for removal of nonaqueous phase liquids from groundwater aquifers, concern over the effects of the solvent on native microorganisms and their ability to degrade residual contaminant has also arisen. This study assessed the impact of ethanol flushing on the numbers and activity potentials of trichloroethylene (TCE)-degrading microbial populations present in aquifer soils taken immediately after and 2 years after ethanol flushing of a former dry cleaners site. Polymerase chain reaction analysis revealed soluble methane monooxygenase genes in methanotrophic enrichments, and 16S rRNA analysis identified Methylocystis parvus with 98% similarity, further indicating the presence of a type II methanotroph. Dissimilatory sulfite reductase genes in sulfate-reducing enrichments prepared were also observed. Ethanol flushing was simulated in columns packed with uncontaminated soils from the dry cleaners site that were dosed with TCE at concentrations observed in the field; after flushing, the columns were subjected to a continuous flow of 500 pore volumes of groundwater per week. Total acridine orange direct cell counts of the flushed and nonflushed soils decreased over the 15-week testing period, but after 5 weeks, the flushed soils maintained higher cell counts than the nonflushed soils. Inhibition of methanogenesis by sulfate reduction was observed in all column soils, as was increasing removal of total methane by soils incubated under methanotrophic conditions. These results showed that impacts of ethanol were not as severe as anticipated and imply that ethanol may mitigate the toxicity of TCE to the microorganisms.