We present a novel approach, based on image analysis and modelling, to study the impact of morphological variability (roughness) and fluid dynamics on substrate mass fluxes in biofilms. Specifically, we used this method to assess substrate fluxes in counter-diffusional autotrophic biofilms in a hydrogen-based membrane biofilm reactor. The physical structure of the biofilm was determined in situ at the meso-scale using stereomicroscopy. Image analysis was used to characterize the biofilm structure, and substrate profiles were obtained using microsensors. A two-dimensional, continuum biofilm model including microbial reactions, mass transport, and fluid dynamics was developed to compute substrate conversion in irregularly shaped counter-diffusional biofilms. Experimental biofilm structures were reproduced in the model and simulated under the prevailing substrate and hydrodynamic conditions for flow velocities varied over three orders of magnitude. Model calculations were consistent with experimental results and showed enhanced conversion rates with increased roughness at higher flow velocities. Also, modelling showed that conversion rates in counter-diffusional biofilms were typically higher than in co-diffusional biofilms. This study highlights the potential to use a simple image acquisition approach coupled to a theoretical model, to evaluate biofilm overall substrate utilization related to biofilm morphological heterogeneity.