Abstract The effects of temporal and spatial changes in biological activity and biomass amount on biofilter performance were investigated in a lab-scale trickle-bed air biofilter at a toluene loading of 46.9 g m −3 h −1 under two different experimental strategies, namely, periodic backwashing at a rate of 1 h once a week and 2 d starvation. Analysis of the overall reaction for toluene metabolism revealed that cell synthesis was relatively favored over toluene oxidation in the inlet section of the biofilter, but over time its oxidation became favored throughout the biofilter bed. Periodic in situ backwashing with media fluidization effectively made even spatial distribution of biomass along the bed media, by which consistent high removal performance in the biofilter has been attained. After 2 d starvation, the ratio of the biofilm EPS to the total biomass increased along the media bed depth, while the total biomass in the media bed subsequently decreased. The presence of sufficient biomass and microbial activity favorably influenced biofilter reacclimation after restart-up following starvation.