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Wound infections are the main complication when treating skin wounds. This work reports a novel antimicrobial material using green synthesized zinc oxide nanoparticles (ZnONPs) incorporated in polymeric fibers for wound healing purposes. ZnONPs are a promising antimicrobial nanomaterial with high activity against a range of microorganisms, including drug-resistant bacteria. The electrospun fibers were obtained using polyacrylic acid (PAA) and polyallylamine hydrochloride (PAH) and were loaded with ZnONPs green synthesized from Ilex paraguariensis leaves with a spherical shape and ~18 nm diameter size. The fibers were produced using the electrospinning technique and SEM images showed a uniform morphology with a diameter of ~230 nm. EDS analysis proved a consistent dispersion of Zn in the fiber mat, however, particle agglomerates with varying sizes were observed. FTIR spectra confirmed the interaction of PAA carboxylic groups with the amine of PAH molecules. Although ZnONPs presented higher antimicrobial activity against S. aureus than E. coli, resazurin viability assay revealed that the PAA/PAH/ZnONPs composite successfully inhibited both bacteria strains growth. Photomicrographs support these results where bacteria clusters were observed only in the control samples. The PAA/PAH/ZnONPs composite developed presents antimicrobial activity and mimics the extracellular matrix morphology of skin tissue, showing potential for wound healing treatments.