This study aimed at evaluating stratification of hydraulic resistance over the depth of membrane biofilms developed during gravity driven membrane (GDM) ultrafiltration. Biofilms were grown in membrane fouling simulators (MFS) for 25 days under 3 contrasting influent conditions: nutrient enriched, phosphorus limiting, and river water influent. Shear rheology was used to determine the biofilms material characteristics (e.g., elasticity, yield stress). Optical coherence tomography (OCT) was used to determine the response of the biofilms physical structure (e.g., thickness) to increased hydraulic shear stress from 0 up to 2.6 Pa. Stratification was observed in nutrient enriched and river water biofilms and which partially detached by erosion of their rough top layer under low shear conditions. A cohesive base layer remained attached to the membrane surface under high shear conditions, indicating stratification in cohesion over the biofilms depth. Erosion of the top layer only resulted in a slight decrease in the biofilm hydraulic resistance indicating that the very cohesive and thin base layer also represent significant hydraulic resistance. Conversely, P limiting biofilms had uniform structural and mechanical characteristics, detaching by sloughing and peeling at low and high shear stress respectively. Biofilms formed under phosphorus limiting conditions had equal elasticity, yet a lower yield stress compared to nutrient enriched and river water biofilms respectively and were more susceptible to detachment. The scientific implication of the presented work demonstrates that biofilm detachment is influenced by both the structural (surface heterogeneity) and material characteristics (e.g., yield stress), with practical relevance for hydraulic resistance during GDM filtration.