In this study, the polyvinyl alcohol (PVA) and sodium caseinate (SC) nanofibers were produced by a single-fluid electrospinning method from their blends. Afterward, the cross-linking process with two different methods was applied to the PVA/SC (70/30, v/v) ratio, which was selected according to the surface and mechanical properties of the electrospun mat. In the first method, different ratios (15%, 20%, 25%, and 30%) of glutaraldehyde (GLA) cross-linking agents were added to the PVA/SC solution and then, PVA/SC/ GLA nanofibers were obtained. In the second method (in-situ method), the nanofibers obtained from the PVA/SC solution were cross-linked by dipping into the cross-linking solution. After, PVA/SC/GLA/Zinc oxide nanoparticles (ZnO NP) mats were obtained by adding ZnO NP at different rates to the PVA/SC/GLA (7030-25GLA) solution, which was chosen according to the results of thermal, mechanical, and moisture test. In addition, performing tests, a cytotoxicity test for fibroblast cell line (L929), and in vitro antibacterial test for Escherichia coli and Staphylococcus aureus were also applied to them.