Biofilm attachment and growth in membrane filtration systems are considerably influenced by the localized flow inside the feed channel. The present work aims to map the biofilm attachment/growth mechanism under varying flow conditions. Effect of varying clearance region (space between the spacer filament and membrane surface) on biofouling pattern is investigated by using three 3D-printed pillar spacers having different filament diameters of 340, 500, and 1000 µm while maintaining the same pillar orientation, diameter and height. Direct Numerical Simulations (DNS) and Optical Coherence Tomography (OCT) were carried out to accurately predict the local hydrodynamics behavior and in-situ monitor the biofilm formation. On spacer filaments, biofouling attachment is primarily observed in the regions where low and non-fluctuating shear stresses are present. Conversely, on membrane surface, highest biofouling attachment was observed under spacer filaments where high shear stresses are prevalent along with low clearance height. Furthermore, as filtration time progresses, the biofilm grows faster on the membrane in the center of spacer cells where low shear stress with steady hydrodynamics conditions are prevalent. The proposed hydrodynamics approach envisages a full spectrum of spacer design constraints that can lead to intrinsic biofilm mitigation while improving filtration performance of membranes based water treatment.