The use of chitosan biofilm in biomedical fields has a vast therapeutic potential due to useful properties such as biodegradability and antimicrobial activity, however, to achieve successful application some physicochemical properties need to be improved. In this context, the addition of glycerol to this biofilm is an alternative to improve its wettability and flexibility. Thus, low and high concentrations of plasticizer were added to this biomaterial for later characterization regarding its wettability, microstructure, mechanical and chemical properties. Glycerol addition to chitosan resulted in biofilms with more homogeneous surfaces, increased wettability, and increased flexibility, without significant changes in its chemical structure.