This research describes the preparation of membranes with chitosan (CS) as the polymeric matrix and cellulose nanocrystals (CNC) as reinforcement. The aim was to evaluate their physical, mechanical and biological properties, and to determine their potential for biomedical use. Membranes were prepared via casting CNC suspensions in CS solution, at CNC concentrations of 0.5%, 1.0% and 2.0% (w/w) with pure chitosan as a reference. Analysis of membrane properties was performed using several techniques, such as ATR-FTIR, SEM, swelling test, maximum water absorption, dynamical mechanical analysis and in vivo (Winstar rats) biocompatibility and biodegradability assays for biological evaluation. Experimental results established that CNC reduced swelling rates and increased the maximum water absorption when CNC concentration was higher. Therefore, the presence of CNC in the matrix reduced Young's modulus by approximately 50% in comparison with pure chitosan. All formulations demonstrated biocompatibility and biodegradability values ranged between 4% and 21% in the 30 days after implantation. Based on these results, these membranes may be of use for biomedical applications.