Methods for characterizing nitrifying bacteria within biofilms are of key importance to understand and optimize the nitrification kinetics of attached growth treatment facilities. In this work, we propose an analytical protocol based upon environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CSLM) in combination with fluorescent in situ hybridization (FISH) to characterize the structure of nitrifying biofilm as it remains attached to the original reactor substratum. This protocol minimizes the loss of mass and distortion of in situ perspective commonly associated with traditionally applied microscopic techniques and thereby enables a more accurate estimation of the nitrifying biomass within biofilm attached to the substratum. The use of ESEM eliminates the destructive preparatory procedures associated with traditional scanning electron microscopy and thus the loss of mass and shrinking of the samples. ESEM is used in this study to evaluate the percent coverage of the substratum with biofilm and the biofilm thickness. CLSM-FISH is used to determine cell counts in the biofilm and to characterize the undisturbed substratum/biofilm interface. By hybridizing and analyzing the nitrifying biofilm using CLSM as it remains attached to the substratum, the loss of material and distortion of in situ perspective associated with the biofilm detachment process is minimized. Moreover, by conducting the CLSM analysis directly on the nitrifying biofilm as it remains attached to the substratum it is shown that cell counts at the substratum/biofilm interface differ significantly from that located above the interface.