Biofilms are surface-attached and matrix-enclosed microbial communities that dominate microbial life in numerous ecosystems. Using flumes and automated optical coherence tomography, we studied the morphogenesis of phototrophic biofilms along a gradient of hydraulic conditions. Compact and coalescent biofilms formed under elevated bed shear stress, whereas protruding clusters separated by troughs formed under reduced shear stress. This morphological differentiation did not linearly follow the hydraulic gradient, but a break point emerged around a shear stress of ~0.08 Pa. While community composition did not differ between high and low shear environments, our results suggest that the morphological differentiation was linked to biomass displacement and reciprocal interactions between the biofilm structure and hydraulics. Mapping oxygen concentrations within and around biofilm structures, we provide empirical evidence for biofilm-induced alterations of oxygen mass transfer. Our findings suggest that architectural plasticity, efficient mass transfer, and resistance to shear stress contribute to the success of phototrophic biofilms.