Silver nanoparticles (AgNPs) are widely explored to enhance the antibacterial property of medical devices such as wound dressings. However, no consensus has been reached on the efficacy and safety of AgNP incorporation. This study aimed (1) to elucidate the effect of proteins and inorganic ions on the antibacterial properties, (2) to confirm the antibacterial efficacy in vivo, and (3) to evaluate the wound healing ability of AgNPs incorporation into chitosan-based membranes. Three membranes with different amount of AgNPs were prepared. The antibacterial properties and silver release profile were evaluated after interacting with phosphate buffered saline or with serum in vitro. The antibacterial efficacy and the wound healing ability were explored in vivo. The results indicated that the biological environment had strong influences on the silver release: the inorganic ions resulted in a slow release whereas the proteins formed a barrier to block the silver release. Consequently, high amount of AgNPs incorporation was necessary to achieve in vivo antibacterial effects. Moreover, the addition of AgNPs did not alter the wound healing rate and tissue response. We can conclude that the AgNP incorporation can enhance antibacterial efficacy of biomaterials without altering the wound healing ability of the chitosan-based membranes.