Packing is the most important factor in biofilter design. A structured mixed packing (SMP) material, combined with various organic and inorganic materials (mineral matter is $80.18{pm}0.48%$, w : w), was constructed by ureaformaldehyde resin in order to minimize the disadvantages of these materials when used as stand-alone components. The performance of the toluene biotrickling filter (BTF) packed with SMP was compared with the other BTFs packed with a ceramic raschig ring, ceramic pall ring, and lava rock, respectively, for 217 day under various operating conditions. Real-time PCR and DGGE techniques were applied to reveal the gene coding for the toluene-degrading enzymes and the bacterial community structure in the BTFs. The toluene-degradation gene copies exponentially increased, and bacterial diversity significantly decreased with the improving elimination capacities of the BTFs. The overload and shutdown operations resulted in insignificant fluctuations in the toluene-degradation gene copies at equal levels as well as a slight variation in the bacterial community structures in the BTFs. Various putative toluene-degrading bacteria were found using sequencing bands from the DGGE gels; some bacteria, such as Burkholderia spp., were further confirmed by real-time PCR; other bacteria, such as Alcaligenes spp., might not have been reported. The packing properties of SMP material supported more toluene-degradation gene copies in the biofilm, and higher toluene-degrading bacterial diversity of the BTF, than did inert packing. Thus, the BTF with SMP demonstrated excellent performance, suggesting the suitability of SMP for real applications, whereas the capabilities of inert packing materials are more suited to the treatment of steady low VOC loads.