Peripheral Nerve Injuries (PNI) have been raising major concerns in regenerative medicine for several years as unsatisfactory recovery continues to be a significant clinical challenge [1]. Chitosan, because of its good biocompatibility and physicochemical properties has been widely used as a biomaterial in tissue engineering (TE) scaffolding [2]. The Degree of Acetylation (DA) is a key parameter that can be controlled and has a major impact on material properties, such as degradation and cell adhesion, with the best results associated to a DA close to 0%, for several TE applications [3, 4]. In this study, chitosan films with three low and different DAs (DA I ~ 1%; DA II ~ 2% and DA III ~ 5%) were produced from Altakitin medical grade chitosan powders. In an effort to ultimately discover the finest chitosan film for Peripheral Nerve Regeneration (PNR), surface and bulk properties of the films were thoroughly investigated by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Contact angle and Surface Energy measurements, FTIR, X-Ray Diffraction (XRD), Differential Scanning Calorimetry (DSC), Degradation and Water Uptake, Bioactivity and Mechanical Properties. Biological assays using L929 cell line and Immortalized Schwann cells were also seeded on the materials in order to assess cell viability, adhesion and proliferation in these membranes. Controlling DA of chitosan films is a promising way to tune material characteristics, even at a molecular level.