It is important to control biofilm cohesiveness to optimize process performance. In this study, a membrane-aerated biofilm reactor inoculated with activated sludge was used to grow mixed-culture biofilms of different ages and thicknesses. The cohesions, or cohesive energy levels per unit volume of biofilm, based on a reproducible method using atomic force microscopy (F. Ahimou, M. J. Semmens, P. J. Novak, and G. Haugstad, Appl. Environ. Microbiol. 73:2897-2904, 2007), were determined at different locations within the depths of the biofilms. In addition, the protein and polysaccharide concentrations within the biofilm depths, as well as the dissolved oxygen (DO) concentration profiles within the biofilms, were measured. It was found that biofilm cohesion increased with depth but not with age. Level of biofilm cohesive energy per unit volume was strongly correlated with biofilm polysaccharide concentration, which increased with depth in the membrane-aerated biofilm. In a 12-day-old biofilm, DO also increased with depth and may therefore be linked to polysaccharide production. In contrast, protein concentration was relatively constant within the biofilm and did not appear to influence cohesion.