Polyelectrolyte layer-by-layer (LbL) nanofilms are interesting polymeric structures, built by alternating adsorption of positively and negatively charged polyelectrolytes. They consist of multilayer sheets with nanometric overall thickness, and they can be used as supports and surface coatings for in vitro and in vivo cell and tissue growth and regeneration. The present study focuses on nanofilms based on alternated layers of poly(sodium-4-sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) fabricated using spin-assisted LbL assembly (SA-LbL). The fabrication process used to assemble polyelectrolyte nanofilms made of up to 60 bilayers is described, and the influence of different surface charges (i.e. changing the terminal layer) and of different film composition (e.g. varying PSS molecular weight) on cell behaviour is investigated. In particular, C2C12 skeletal muscle cells' viability, proliferation and differentiation on six different typologies of polyelectrolyte nanofilms are evaluated and quantified, giving a reference for skeletal muscle regeneration capabilities on such kind of structures.