Three types of multilayer films made from poly(L-lysine)/hyaluronan, chitosan/hyaluronan, and poly(allylamine hydrochloride)/poly(L-glutamic acid), were used to investigate the interplay between film mechano–chemical properties and cell adhesion. We showed that C2C12 myoblast adhesion and proliferation depended on the extent of film cross-linking for all films whatever their internal chemistry. Cell spreading areas were found to correlate with the film's stiffness and to be distributed over a unique curve. Immuno-staining of the cytoskeletal components revealed the formation of F-actin stress fibers and vinculin plaques only on stiff films. Finally, we compared our results with previous studies performed on polyacrylamide and PDMS gels, two recognized materials for mechano-sensitivity studies. We found that the effect of substrate stiffness on cell spreading is material-dependent.