Biofilters for the removal of methane using tobermolite, perlite and polyurethane as packing materials have been undergoing recent development. The effects of these packing materials on methane oxidation activity were evaluated in this study. Mixed methanotrophs (consortia A, B, C and D) from wetland and landfill soils were used as the inoculum sources. The influences of packing materials, consisting of tobermolite, perlite, and polyurethane, on the methane oxidation rate and methanotrophic biomass, were estimated. When perlite was added into the methanotrophic cultures, the methane oxidation rate was more than twice that of the control (without packing materials), and the methanotrophic biomass increased more than 10 fold. The ratio of methanotrophic bacteria to total bacteria under with tobermolite packing material was higher than the control and the other packing materials, indicating that tobermolite can serve as a specific packing material where dominance of methanotrophs is desired. Therefore, perlite and tobermolite provide habitats which increase the activity of methanotrophic bacteria, and these packing materials are promising for use in methane oxidation processes.