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The biological use of nanocomposites (NCs) is highly intriguing and is more acknowledged for its value, particularly in nanomedicine. The key goal of this study is to investigate the influence of vanadium nanoparticles (VNPs) addition on PVA’s morphological, optical, and electrical properties. In this work, the PVA/V nanocomposites (NCs) were fabricated with different VNP contents of (0.02, 0.04, and 0.06) wt.% by the casting solution technique. The optical microscopy (OM) and scanning electron microscope (SEM) have been used to examine the surface morphological features of produced films, which showed that the VNP clusters were well dispersed in the polymer medium. The intermolecular interaction of the PVA/V nanocomposite was studied by FTIR examination. The interaction between PVA and V was confirmed by observing the change in IR absorption intensity. The UV–Vis approach was used to explore the optical characteristics of the UV–Vis region. The experimental data demonstrate that variations in the concentration of VNPs significantly impact the absorption. For the doped samples, the absorbance against wavelength is exponential. There is evidence of interface between polymer and nanoparticles because the optical band gap has shrunk from 4.50 eV to 3.30 eV, with a corresponding narrowing of the gap between the valence and conduction bands. The electrical characteristics of alternating current were investigated in the frequency range of 100–5 MHz. The dielectric constant and loss of NC films reduced as the concentration of VNPs rose, whereas electrical conductivity increased. With rising VNPs, the attenuation coefficient values rose. The inhibition zone diameters of Staphylococcus aureus bacteria increased with the increase of VNP contents. We showed that adding polyvinyl alcohol (PVA) to V composites increases their antimicrobial (ANB) activities. Based on these findings, NC films can be used for gamma-ray protection and antimicrobial (ANB) purposes.