Methyl tertiary-butyl ether (MTBE) is a synthetic chemical used in unleaded gasoline as an additive to reduce levels of ozone and carbon monoxide from auto exhaust. Due to its chemical and recalcitrant properties, MTBE has caused groundwater contamination worldwide. A laboratory-scale biofilter made of a natural fiber (kenaf) mat and inoculated with MTBE-degrading microorganisms, was evaluated for MTBE removal efficiency. Operational parameters of oxygen flow rate, hydraulic retention time (HRT), yeast extract and initial MTBE concentration were varied and MTBE removal efficiencies determined. Four kinetic models were evaluated to describe the MTBE removal in the reactor. Formaldehyde and tertiary butyl alcohol (the most two reported MTBE biodegradation byproducts) were not found in the effluent; instead, carbon dioxide was monitored as the end product based on the results of a metabolic mass balance evaluation. Toxicity of treated effluent was evaluated by employing the Microtox acute toxicity test and comparing that to the influent.