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Abstract Zinc Oxide (ZnO), a metal oxide semiconductor, is well known for its distinctive optical, morphological, catalytic, and sensing characteristics. Incorporation of polyaniline (PANI) into ZnO enhances its microstructural, optical, and morphological characteristics. The present study focuses on synthesizing PANI/ZnO-based nanocomposites (NCs). PANI was synthesized via in situ chemical oxidative polymerization of aniline in an acidic medium using ammonium persulfate (APS) as the oxidant. Subsequently, PANI/ZnO nanocomposites with varying PANI loadings (1, 3, 5, and 10 wt.%) were synthesized through a facile co-precipitation method. The synthesized nanocomposites were systematically characterized using X-ray Diffraction (XRD), Photoluminescence Spectroscopy (PL), X-ray Photoelectron Spectroscopy (XPS), Fourier Transform Infrared Spectroscopy (FTIR), Raman Spectroscopy, Transmission Electron Microscopy (TEM), Field Emission Scanning Electron Microscopy (FESEM), and Impedance Spectroscopy. XRD analysis confirmed the formation of the hexagonal wurtzite crystal structure with lattice distortion, with crystallite sizes ranging from ~21.23 to ~30.31 nm. Morphological studies using TEM and FESEM analysis confirmed the uniform dispersion of PANI over the surface of ZnO nanoparticles. TEM analysis revealed particle sizes in the range of ~26.23-36.23 nm. FTIR, PL, XPS, and Raman studies provided additional evidence of strong chemical interaction between PANI and ZnO. Dielectric analysis reveals strong interfacial interactions between PANI and ZnO, indicating interfacial polarization and improved charge transport behavior. The antibacterial activities of the synthesized nanocomposites were carried out against both Gram-positive and Gram-negative bacteria, indicating their potential as effective antimicrobial agents. The PANI/ZnO nanocomposites provide a sustainable material system due to their low-cost synthesis, environmental stability, and the use of non-toxic, widely available components, making it a green alternative for antibacterial applications.