In industrial activities, operation of air biofilters may be interrupted for a long period of time. In order to examine the effect of shut-down on the performance of biofilters, a pilot scale biofiltration unit was restarted up after a non-use duration of eight months. After a reacclimation period of eight days, the biofilter reached an elimination capacity of 35 g.m−3.h−1 compared to 38 g.m−3.h−1 before shut-down, for the same operating conditions. The results obtained indicate that the microorganism activity was completely restored and this was confirmed by the microbial count tests performed on filter bed material samples. The impact of varying the inlet concentration of toluene on biofilter performance was also investigated. A maximum elimination capacity of 120 g.m−3.h−1 was achieved, compared to 135 g.m−3.h−1 before shut-down. The biofilter removal efficiency exceeded 96% for inlet concentrations up to 1.8 g.m−3. Temperature measurements revealed that the filter bed temperature depends strongly on the intensity of microbial activity and the biofilter effectiveness in eliminating toluene. Experimental data were compared with the calculations of a mathematical model. Comparison showed that the theoretical model was successful in predicting the performance of the biofiltration unit.