We report on the preparation of layer-by-layer (LbL) assemblies based on anionic hydrophobically modified polysaccharides and different polyeations. We systematically investigate the influence of the effect of the hydrophobicity of the polysaccharide derivatives on the multilayer buildup. We provide clear evidence that the presence of hydrophobic interactions contributes strongly to stabilize the adsorbed layers. In addition, the correlation between the study of the multilayer growth and of the solutions properties of the amphiphilic derivatives shows that the thickness of adsorbed layers is directly determined by the conformation of amphiphilic chains in solution. These results are in agreement with the presence of hydrophobic microdomains in the films.