Membrane fouling is the major problem that hinders the practical application of membrane bioreactor (MBR) systems. Since the MBR system includes living microorganisms and their metabolites, the fouling mechanism is even more complex than that of conventional membrane separation processes. This paper provides an improved understanding of the influence of filamentous bacteria on membrane fouling in microfiltration of activated sludge wastewater. In order to make clear the membrane fouling mechanism of filamentous bacteria, the physicochemical characteristics of sludge flocs, including extracellular polymeric substance (EPS), zeta potential, relative hydrophobicity (RH) and floc morphology were systematically investigated. The results showed that the sludge flocs with negligible filamentous bacteria led to severe membrane pore blocking, and the sludge flocs with filamentous bacteria created the formation of a non-porous cake layer on the membrane surface. The excess growth of filamentous bacteria resulted in much more release of EPS, lower zeta potential, higher hydrophobicity of sludge flocs and more irregularly shaped flocs, which did great harm to membrane filtration. The sludge flocs with a small quantity of filamentous bacteria had a positive effect on membrane permeation. It is important to control filamentous bacteria concentration in the operation of MBRs.