We report on the preparation of polyelectrolyte films based on biopolymers whose nanomechanical properties can be tuned by photo-cross-linking. Cationic poly(L-lysine) was layer-by-layer assembled with anionic hyaluronan (HA) derivatives modified by photoreactive vinylbenzyl (VB) groups. The study of the multilayer buildup by quartz crystal microbalance with dissipation monitoring showed that the presence of VB groups does not influence significantly the multilayer growth. Then the VB-modified HA incorporated into the films was cross-linked upon UV irradiation. UV spectroscopy measurements showed that the cross-linking rate of the multilayers increases with the amount of VB groups grafted onto HA chains. Force measurements performed by atomic force microscopy with a colloidal probe proved that the rigidity of the cross-linked films increases with the grafting degree of HA chains and consequently the number of cross-links. Cell culture assays performed on non-cross-linked and photo-cross-linked films with myoblast cells demonstrated that cell adhesion and proliferation are considerably improved with increasing film rigidity.