Biofilms are inevitable and typically adhere on solid surfaces such as the membrane surfaces in membrane bioreactors. Aeration has been widely used in the aerobic stage of water treatment to hydrodynamically generate shear stress to disrupt biofilm formation. The current study quantified the influence of shear stress on mature biofilms by examining its influence on bacteria Quorum Sensing behaviour which involves c-di-GMP molecules. The level of external shear stress was determined via validated Computational Fluid Dynamics simulation and the level of c-di-GMP was measured directly. It was found that bacteria are very sensitive to environmental shear stress; a small change of 0.05 Pa can lead to an intracellular c-di-GMP fluctuation of 5.6 pmol/mg protein. The mediating effect of shear stress on c-di-GMP level is nonlinear. In MBR systems, a time-averaged shear stress exceeding 0.72 Pa is indicated as it generates lower c-di-GMP and a looser biofilm that has low biodiversity.