In this work, chitosan-based films containing gelatin and chondroitin-4-sulfate (C4S) with and without ZnO particles were produced and tested in vitro to investigate their potential wound healing properties. Chitosans were produced from shrimp-head processing waste by alkaline deacetylation of chitin to obtain chitosans differing in molecular weight and degree of deacetylation (80 +/- 0.5%). The film-forming solutions (chitosan, C4S and gelatin) and ZnO suspension showed no toxicity towards fibroblasts or keratinocytes. Chitosan was able to agglutinate red blood cells, and film-forming solutions induced no hemolysis. Film components were released into solution when incubated in PBS as demonstrated by protein and sugar determination. These data suggest that a stable, chitosan-based film with low toxicity and an ability to release components would be able to establish a biocompatible microenvironment for cell growth. Chitosan-based films significantly increased the percentage of wound healing (wound contraction from 65 to 86%) in skin with full-thickness excision when compared with control (51%), after 6 days. Moreover, histological analysis showed increased granulation tissue in chitosan and chitosan/gelatin/C4S/ZnO films. Chitosan-based biopolymer composites could be used for improved biomedical applications such as wound dressings, giving them enhanced properties.