Bacillus subtilis broths under different culture times are filtered in a cross-flow microfilter. The operating condition effects, such as cross-flow velocity, transmembrane pressure, and broth culture time, on the filtration flux, cake properties, and extracellular polymeric substances (EPS) transmissions are discussed thoroughly. The culture broths contain B. subtilis cells and EPS which is characterized as polysaccharides (hydrocarbons) and proteins. An increase in broth culture time leads to higher concentrations of cells, soluble and extractable EPS. The total protein to polysaccharide concentration ratio in the broths is ca 0.2. However, the soluble polysaccharide concentration is 10-fold higher than that of soluble proteins. The filtration flux increases with increasing cross-flow velocity or transmembrane pressure. However, the impact of cross-flow velocity is more significant. The filter cake resistance formed by B. subtilis cells and EPS flocs plays the most important role in determining the overall filtration resistance. The mass and average specific filtration resistance of cake can be estimated using a force balance model and empirical equations. The cake structure and thickness are analyzed using SEM. A thicker and more compact cake may be formed under longer broth culture time. Most soluble polysaccharide and protein molecules have the opportunity to penetrate through the cake and membrane into the filtrate because the solute transmissions are measured as high as 0.75-1.0. The influences of operating conditions on the polysaccharide and protein transmissions are negligible. Therefore, to enhance filtration flux by increasing transmembrane pressure or cross-flow velocity is beneficial to improve separation efficiency, especially by increasing cross-flow velocity.