Microbial remediation of methyl tert-butyl ether-contaminated aquifers has been widely studied since their cost/efficiency ratios are lower than other remediating techniques. Based on previous studies, simultaneous assessment of two inocula (Acinetobacter calcoaceticus M10 and a co-culture between strain M10 and Rhodococcus ruber E10) was performed for methyl tert-butyl ether bioremediation in a designed pilot-scale biofilter. A non-inoculated biofilter was included for comparison. Temporary bioremediation of methyl tert-butyl ether (up to 80 % removal) from the biofilter inoculated with the consortium was observed within 44 days. In addition, the taxonomic profile (temporal temperature gradient gel electrophoresis from RNA extracts) from the latter biofilter contained more active strains than in the other two systems. The presence of strains M10 and E10 in temporal temperature gradient gel electrophoresis analysis from RNA extracts and high presence of strain E10 in temporal temperature gradient gel electrophoresis analysis of DNA extracts, along with alkB amplification of both strains in the biofilter, suggest that the co-culture inoculum was responsible for the methyl tert-butyl ether removal.