One of the complication of diabetes mellitus is chronic wounds. The healing of wounds in diabetic patients is retarded by the elevation in the pro-inflammatory cytokines secretion and free radicles accumulation. Wound management in diabetic patients requires preventing bacterial biofilm development. Due to the wound healing activity of chitosan (CS), lecithin (Le) and melatonin (M), the present study aimed to load melatonin on CS/Le NPs and examine their effect on diabetic wounds infected with Staphylococcus aureus. Melatonin loaded chitosan/lecithin nanoparticles (M-CS/Le NPs) were physically characterized and their antioxidant, anti-inflammatory and antimicrobial activities were examined in vitro. Male Sprague Dawley rats included two division (non-diabetic and diabetic) which were further divided in nine groups. Diabetes induction and follow up throughout the experimental period was confirmed by measuring the levels of fructosamine and blood glucose. Full-thickness wounds was induced in both non-diabetic and diabetic animals followed by infection with Staphylococcus aureus according to the experimental design. The wound healing effect of M-CS/Le NPs was evaluated through measurements of the oxidative stress, inflammatory cytokines and apoptotic proteins. Our results showed the anti-microbial, free radical scavenging and hemolysis inhibition effects of M-CS/Le NPs in vitro. Moreover, the preparation of M-CS/Le NPs decreased the dose of used melatonin (when compared to free melatonin). M-CS/Le NPs significantly decreased the wound area percent in treated infected wounds of both non-diabetic and diabetic rats more than free melatonin or unloaded CS/Le NPs. In conclusion, M-CS/Le NPs promoted the wound healing in Staphylococcus aureus-infected wounds in diabetic rats.