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Skin is the largest protective organ that could be recurrently wounded and attacked by microorganisms. The wounded skin safeguarding and supporting were intended through natural derivatives. Fish collagen (Cg) type I, extracted from sea bream (Spondyliosoma cantharus), chitosan nanoparticles (NCht) from shrimp shells, and henna (Lawsonia inermis L.) leaves extract (He) were produced and physiochemically characterized. The antimicrobial potentialities of these compounds and their composites were assessed toward skin pathogens (Candida albicans and Staphylococcus aureus) using various assaying methods and microimaging techniques. The infrared and electrophoretic analysis of Cg validated its characteristics, and the IR-spectroscopic analysis of the compounds/composites indicated their physiochemical attributes and interrelations. The produced NCht particles had a diameter range of 64.6-308.8 nm, 104 nm mean diameter, and +31.3 mV zeta potentiality. Both NCht, He, and NCht/He composite exhibited significant antimicrobial potentiality toward skin pathogens; NCht/He was the strongest with inhibitory concentrations of 20.0 and 22.5 μg/mL and inhibition zones of 25.7 and 26.8 mm against S. aureus and C. albicans, respectively. The electron micrographs verified the synergistic microbicidal action of NCht/He, as they led to severe microbial lysis and deformations. The skin wounds’ treatment with NCht/He/Cg composite promoted the fastest and complete healing of wounded rats’ skin during 8 days of local treatment, with the absence of inflammation and infection signs; treated with NCht/He/Cg composite, the wound area vastly reduced from 63.6 mm2 to 15.9 and 9.1 mm2 after 2 and 4 days, respectively. The natural NCht/He/Cg composites are recommended as topical applications for optimum skin disinfection and regeneration.