Deep bed filtration is widely applied in bioprocessing, virus filtration, and water purification to remove small impurities, such as particles or virus fragments. However, more needs to be understood about the parameters that influence particle capture and deposition. Detailed simulations and microfluidic filtration experiments with straight pores have been widely investigated, yet realistic membrane porosity features such as pore gradients and roughness have not been addressed in detail. The internal membrane morphology is assumed to have a strong effect on filtration performance. Therefore, this study introduces a new microfluidic membrane mimicking device (MMD) and methodology that analyzes spatio-temporal particle collections in a deep bed filtration MMD with gradually decreasing pore size toward the retentate side. The design allows us to systematically tailor an internal filter surface roughness to investigate its influence on differently sized soft particles and the consequences for filtration performance, in particular, particle capture. Optical investigation and quantitative evaluation show enhanced particle–pillar interactions for increased roughness. This results in a reduced penetration depth and reduced particle breakthrough. The modes of capture or filtration phenomena, such as pore blocking, bridging, or dendrite formation, differ significantly between smooth and rough membrane filter structures. In the case of rough inner filter surfaces, those phenomena lead to a less distinct decrease in volumetric flow, allowing a longer filtration process. The microfluidic study offers a detailed investigation of how microscale phenomena influence the filtration process. The results provide a fundamental understanding of microscale filtration effects based on roughness and, hence, motivate a tailoring of the internal membrane filter surface according to the filtration problem at hand.