The objective of this study was to use indirect methods to assess the biofilm cohesion and its vertical stratification. Biofilms were grown under low hydrodynamic strengths in two reactors, a low shear stress reactor (LSSR) or under a defined shear stress in a Couette-Taylor Reactor (CTR), using different electron donors and acceptors. The stratification of biofilm cohesion was characterized from a gradual increase of the hydrodynamic strengths in terms of shear stress (erosion) and abrasion (collisions of particles). Whatever the nature of the biofilms, erosion tests demonstrated a gradual impact of a wall shear stress increase on the biofilms remaining on the substratum surface. These observations demonstrated that the biofilm cohesion was heterogeneous and increased with the biofilm depth. Both erosion and abrasion tests highlighted a basal zone of high cohesion. Investigations based on PCR-SCCP and oxygen uptake rate showed the presence of active microorganisms in this zone.