This study reported the fabrication and characterization of an electrospun wound dressing composed of polyvinyl alcohol (PVA), nanochitosan (NC), and Artemisia ciniformis extract (AE). NC and AE wt% were defined as process parameters, and their effects on the diameter, mechanical properties, and cell attachment of the fibers were investigated using the Design Expert 7.0 software. The software provided quadratic equations, which were investigated statistically and graphically, confirmed that with the increase in the NC portion in the samples, the mechanical strength and fiber diameter increased. The increase is probably due to the proper dispersion of nanoparticle into a PVA matrix and the increased viscosity of the solution; however, cell viability showed an increasing/decreasing trend. The presence of AE lowered the mechanical properties of the samples while positively affecting cell viability. Furthermore, the samples had significant antibacterial properties against Escherichia coli as Gram-negative bacteria and Staphylococcus aureus as Gram-positive one, a notable property for wound dressings. Overall, our findings showed that PVA/NC/AE has an excellent potential for use in biomedical fields, particularly as a wound dressing.