Wound problems in chronic diseases still have imposed a serious financial and emotional burden on the health system and many people in society, and efforts have been made to make a suitable wound dressing. In this study, we fabricated Zein nanofibers containing 1%, 3%, and 5% cerium oxide nanoparticles (Zein/CeNP1%, 3%, and 5% NFs) by electrospinning method. Physicochemical properties of nanofibers studied by FESEM, XRD, FTIR, mechanical properties, release and encapsulation efficiency, WVTR and WAC% on nanofibers. We also evaluated cytotoxicity, internal ROS, and anti-bacterial properties utilizing MTT, DCFH-DA, and agar diffusion methods. The results showed that the synthesized nanofibers have a smooth and bead-free morphology, which have higher diameter and porosity with the increase in the percentage of CeNPs. The nanoparticles have preserved their crystalline form with no chemical bonds in the nanofibers. Zein/CeNP 1% NF has shown better mechanical properties, which is probably due to increase in the number of nanofibers per unit area. From the nanofibers, CeNP has been released rapidly in the first 2 h and then followed with a sustained release. WVTR and WAC% of Zein/CeNP NFs have been significantly higher than Zein NF. Compared to Zein/CeNP 3%, and 5% NF, Zein/ CeNP 1% NF significantly have reduced internal ROS and increase the proliferation of HDF cells. The results have displayed Zein and Zein/CeNPs 1%, 3%, and 5% NFs have not shown any inhibition of growth zone on Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. These results show that Zein/CeNP 1% NF have better physicochemical and biological properties which could be evaluated for wound healing studies.