The biofilm in a full-scale nitrifying trickling filter (NTF) treating municipal wastewater has been investigated with microbiological methods using fluorescence in situ hybridization (FISH) with 16S rRNA oligonucleotide probes in combination with confocal laser scanning microscopy (CLSM) and mathematical modeling using a dynamic multi-species biofilm reactor model. Ammonia oxidizing bacteria (AOB) were found to belong to the genus Nitrosomonas at different depths in the NTF at every sampling occasion, corresponding to different long-term operational conditions for the NTF. Both the measurements and the corresponding simulated predictions showed the same general trend of a decrease with filter depth of the amount of biofilm, the proportion of AOB to all bacteria and the total amount of AOB. The latter decreased by several times from top to bottom of the NTF. Measurements and simulations of potential ammonium oxidizing activity in the biofilm also showed a decreasing activity with depth in the NTF, which generally was operating at close to complete nitrification. However, no difference was observed when the activity was normalized to the amount of biofilm, despite decreasing proportions of AOB to all bacteria with depth in the NTF. This could be explained by diffusion limitations in the biofilm from the upper parts of the NTF according to the biofilm reactor model. The relatively good agreement between the simulations and the measurements shows that the kind of biofilm reactor model used can qualitatively describe an averaged behavior and averaged composition of the biofilm in the reactor.