A novel heterogeneous respirometer for in situ assessment of the biological activity and mass transport phenomena of biofilm developed on packing materials of gas-phase biological filters is presented. The flexible respirometer configuration allows reproducing the operational features of biofilters and biotrickling filters to obtain reliable diagnoses of the bioreactor performance. A batch-operating mode was chosen for the biological assessment in which dynamic concentrations of oxygen, pollutant, and carbon dioxide were online recorded and used to calibrate a mathematical model that considers the principal phenomena occurring in the respirometer. The maximum elimination capacity of two different packing materials colonized by sulfide-oxidizing bacteria and toluene-oxidizing fungal populations was estimated and compared with previously reported values. Additionally the rate controlling phenomena were analyzed using the biofilm effectiveness factor. The experimental system can be easily applied to pilot- and industrial-scale gas-phase biological filters when the kinetic expression of pollutant degradation is available.