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Morphological parameters are commonly used to predict transport and metabolic kinetics in biofilms. Yet, quantification of biofilm morphology remains challenging because of imaging technology limitations and lack of robust analytical approaches. We present a novel set of imaging and image analysis techniques to estimate internal porosity, pore size distributions, and pore network connectivity to a depth of 1 mm at a resolution of 10 µm in a biofilm exhibiting both heterotrophic and nitrifying activities. Optical coherence tomography (OCT) scans revealed an extensive pore network with diameters as large as 110 µm directly connected to the biofilm surface and surrounding fluid. Thin‐section fluorescence in situ hybridization microscopy revealed that ammonia‐oxidizing bacteria (AOB) distributed through the entire thickness of the biofilm. AOB were particularly concentrated in the biofilm around internal pores …