This work presents a comprehensive investigation into the synthesis and characterization of polyvinyl alcohol (PVA) nanofibers modified to enhance antimicrobial efficacy through glutaraldehyde crosslinking and the incorporation of silver nanoparticles. The nanofibers were synthesized using the electrospinning technique, followed by a crosslinking process employing the vapor chamber method with glutaraldehyde/HCl solvent evaporation for 24 h, resulting in a nanofiber mat resistant to water. The introduction of silver nanoparticles was achieved via the chemical reduction method using NaBH₄ as a reducing agent, yielding nanoparticles with a size distribution ranging from 5 to 9 nm and uniformly dispersed within the crosslinked nanofiber matrix. The antimicrobial activity of the resulting composite nanofiber mat was thoroughly evaluated, revealing significantly improved efficacy against a range of microbial pathogens. The mechanisms underlying the enhanced antimicrobial activity, attributed to the synergistic effects of crosslinking and silver nanoparticle incorporation, are discussed in detail. Moreover, the physicochemical properties of the nanofiber mat, including morphology, structure, and composition, were analyzed using various characterization techniques such as SEM, STEM, FTIR, Raman, and EDS. The findings elucidate the potential of this approach for developing advanced antimicrobial materials applicable in diverse fields, including biomedical textiles, wound dressings, and medical devices. This study contributes to the ongoing efforts to combat antimicrobial resistance and improve infection control strategies in healthcare and other relevant sectors.