Recently, nanocomposites produced from clays and metals coated with chitosan have shown wound healing activity. This study aimed to synthesize the zinc oxide/malachite nanocomposite (ZnO/Mlt-NC) and its coating form with chitosan (ZnO/Mlt/Chsn-NC). Physicochemical characterization of the produced nanocomposites was investigated. Biomedical effects of nanocomposites, such as in vivo and in vitro antibacterial activity, antioxidant properties, cytotoxicity, and modulation in the gene expressions of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) and histopathological parameters, were also investigated. Expression intensities of basic fibroblast growth factor (bFGF) and tumor necrosis factor alpha (TNF-α) were also investigated by immunofluorescence staining. To investigate biomedical effects under in vivo conditions, infected wounds were induced and inoculated with Staphylococcus aureus (ATCC 25923), and Pseudomonas aeruginosa (ATCC 27853). The results indicated spherical ZnO nanoparticles on the surface of malachite and strong antibacterial activity and antioxidant properties. The ointments produced from the nanocomposites also exhibited wound healing activity. The administration of the ointments prepared from ZnO/Mlt, and ZnO/Mlt/Chsn NCs decreased the expressions of IL-1β, IL-6, and TNF-α, while it increased the expressions of IL-10, TGF-β and bFGF. In sum, the nanocomposites produced from ZnO, malachite, and chitosan had better biological activity than ZnO/Malachite nanocomposites. We suggest applying ZnO/Mlt/Chsn nanocomposites in the structure of ointments to treat infected wounds after future clinical studies.