Abstract: The goals of the study were to determine the effectiveness of a laboratory-scale biofilter on the removal of methyl tert-butyl ether (MTBE) and investigate the operating parameter effects on biofilter performance. The experimental results show that average MTBE removals of 53.6–93.2% were observed at loads of 2.5–20.1 gm−3 h−1 and an empty-bed residence time of three minutes, after continuous operation for four months throughout the biofilter acclimation period. After a one-day recovery period operation, the biofilter system recovered from the introduction of a shock load. More than 99% removal efficiencies were achieved for the inlet MTBE concentration at 50 ppmv and with the highest residence time. MTBE removals at the bottom section of the biofilter were consistently lower than for the top section, which was attributed to insufficient microorganism growth in the bottom section. The parameters estimated by using the Michaelis-Menten equation were 1.116 ± 0.51 ppmv s−1 for the maximum removal rate (V m ), and 26.38 ± 17.21 ppmv for the half-saturation constant (Ks), evaluated at the biofilter exit.