This article demonstrates the possibility of tuning the degradability of polysaccharide multilayer films in vitro and in vivo. Chitosan and hyaluronan multilayer films (CHI/HA) were either native or crosslinked using a water soluble carbodiimide, 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC) at various concentrations in combination with N-hydroxysulfosuccinimide. The in-vitro degradation of the films in contact with lysozyme and hyaluronidase was followed by quartz crystal microbalance measurements, fluorimetry, and confocal laser scanning microscopy after labeling of the chitosan with fluorescein isothiocyanate (CHIFITC). The native films were subjected to degradation by these enzymes, and the crosslinked films were more resistant to enzymatic degradation. Films made of chitosan of medium molecular weight were more resistant than films made of chitosan-oligosaccharides. The films were also brought in contact with plasma, which induced a change in film structure for the native film but did not have any effect on the crosslinked film. The in-vitro study shows that macrophages can degrade all types of films and internalize the chitosan. The in-vivo degradation of the films implanted in mouse peritoneal cavity for a week again showed an almost complete degradation of the native films, whereas the crosslinked films were only partially degraded. Taken together, these results suggest that polysaccharide multilayer films are of potential interest for in-vivo applications as biodegradable coatings, and that degradation can be tuned by using chitosan of different molecular weights and by controlling film crosslinking.