A heterotrophic biofilm (B1) and a mixed autotrophic-heterotrophic biofilm (B2) were developed in an annular reactor and submitted to an erosion test in order to selectively detach top layers from the bottom layers. Densities of the basal layers were 5-fold higher and 3-fold higher than the densities of the entire biofilms B1 and B2, respectively. After extraction, EPS content in B1 biofilm was found higher in the basal layer (95 mg g(-1) VSS) compared to the top layer (30 mg g(-1) VSS), while B2 biofilm had a higher EPS content in the top layer (303 mg g(-1) VSS) compared to the basal layer (135 mg g(-1) VSS). Hydrophobic Interaction Chromatography (HIC) indicates that hydrophobic EPS (HEPS) in both biofilms reached 21% of EPS in basal cohesive layers, and remained slightly lower or identical (16-19%) in top detached biofilm layers. Strong interacting HEPS were found in a higher proportion in the mixed autotrophic-heterotrophic B2 which was also more diversified in terms of bacterial populations than the B1 heterotrophic biofilm. These results show that HEPS content correlates better with cohesive properties of the biofilm layers than global EPS content and that strong hydrophobic adhesion forces may be related to microbial populations such as the presence of nitrifiers.