The skin, which is a protective barrier for the human body, can be injured from time to time as a result of disease or trauma. Electrospun nanofiber wound dressings made from biopolymers for skin regeneration and wound healing have been widely investigated in recent years. This study successfully prepared PVA-based nanofiber wound dressings loaded with nicotine and quercetin by the electrospinning method to accelerate wound healing. The nanofibers were characterized morphologically, physically, and chemically, and then their cytotoxicity levels and effects on wound healing were evaluated in vitro. First, three different concentrations of PVA solutions were prepared, and one nanofiber wound dressing was produced with each of them. Next, characterization studies of the nanofiber wound dressings were performed, and the optimal formulation was selected. Morphological examinations by SEM analysis revealed that homogeneous and uniform nanofibers with an average diameter of 122.11 ± 36.22 nm and no bead formation were successfully produced. The structural properties of the nanofiber wound dressings were evaluated by DSC and FTIR analyses, which revealed that there was no significant physico-chemical interaction between the polymer and the loaded drugs. In terms of drug loading efficiency, the loading efficiency of nicotine was found to be higher compared to quercetin (quercetin: 21.86 ± 2.1%; nicotine: 99.43 ± 6.1%), and it was also determined that the nanofiber structure produced increased the solubility of quercetin by approximately two times. The results of MTT assay on L929 cells revealed that the prepared nanofiber wound dressings did not show cytotoxicity, on the contrary, they supported biocompatibility by increasing cell viability. The wound-healing process was evaluated using an in vitro scratch test. The F2 nanofiber wound dressing produced approximately a 3-fold increase in wound closure compared to the control. Studies have shown that nicotine and quercetin-loaded PVA nanofiber dressings have the potential to accelerate wound healing.