Microbial infections are considered as one of the most important concerns of the world community. Developing drug delivery systems based on formulation of nanoparticles with antimicrobial agents have shown beneficial effectiveness against microbial infections and related antimicrobial resistance. In this study we prepared and characterized a chitosan based hydrogel loaded with zinc oxide nanoparticles for controlling the release of vancomycin and also improving its antibacterial effect. Characterization studies demonstrated that the developed biopolymeric hydrogel was able to sustained and controlled the release of vancomycin in response to acidic media for 96 hours. Furthermore, antimicrobial studies showed siginificant and efficient antibacterial activity of prepared hydrogel against S. aureus and P. aeruginosa. Based on obtained results, it can be concluded that the prepared chitosan hydrogel containing ZnO nanoparticles has a desirable activity for controlling the release of vancomycin and improving its antibacterial properties.