Recent studies on membrane fouling have made considerable progress in reducing its adverse effects. However, a lack of comprehensive studies focusing on the underlying fouling mechanisms remains. This work aims to address a part of this gap by investigating the influence of feed suspension chemistry and operating conditions on the fouling characteristics of microcrystalline cellulose. Fluid dynamic gauging (FDG) was employed to monitor the properties of fouling layers under varied conditions. FDG results revealed that the cohesive strength of fouling layers increased in the direction towards the membrane, which can be associated with the higher compressive pressures exerted on foulants deposited near the surface. At lower pHs and higher ionic strengths, reduced electrostatic repulsions between particles likely resulted in particle agglomeration, leading to the formation of thicker cakes. In addition, thicker cake layers were also observed at higher feed concentrations, higher operating transmembrane pressures, and longer filtration times. The cross-flow velocity influenced the resilience of fouling layers significantly, resulting in thinner yet stronger cake layers in the transition and turbulent flow regimes. These findings regarding the influence of feed characteristics and operating conditions on the fouling behavior can be beneficial in developing effective antifouling strategies in membrane separation processes.