In the present study, a Quality by Design (QbD) approach was applied in order to develop and optimize a chitosan film formulation loaded with bioactive compounds, intended for further use as an aid in the acceleration of wound healing. Chitosan was chosen as film forming polymer, polyethylene glycol was used as plasticizer, while polyvinyl alcohol was added to improve the bioadhesive properties of the films. Risk assessment strategy was applied to identify the critical formulation variables, which were further introduced as factors of a 3 level Box Behnken design of experiments. The optimal film formulation registered a film thickness of 0.09 mm, solubility in water of 44.9% and a high swelling degree of 2157.4%. The optimized formulation, as well as a placebo formulation without bioactive compounds proved to have a good antibacterial activity. We conclude that in the current study, the QbD approach was successfully applied in order to ensure a good understanding of the manufacturing process, as well as to optimize the formulation of chitosan films. To the best of our knowledge, this is the first time when a QbD approach has been applied in order to develop and optimize a film formulation as a novel wound dressing material.