This study investigates electrospun nanofibers based on polyvinyl alcohol (PVA) and frankincense (Boswellia resin) for potential skin substitution applications. Pure PVA and PVA/frankincense blends were fabricated via electrospinning and characterized for fiber morphology, surface wettability, mechanical properties, and antibacterial activity against Staphylococcus aureus. Scanning electron microscopy (SEM) revealed that pure PVA produced nanofibers with an average diameter of 187.1 ± 21 nm. The incorporation of frankincense increased the fiber diameter to 321 ± 33 nm. Contact angle measurements demonstrated that both pure PVA (30.76°) and the PVA/frankincense blend (30.42°) exhibited hydrophilic surfaces, ensuring consistent wound fluid absorption essential for skin substitute function. Tensile testing showed that the PVA/frankincense blend exhibited increased stiffness, with an E-modulus of 13.38 MPa compared to 8.20 MPa for pure PVA (63% increase), indicating hydrogen bonding interactions between PVA and frankincense components. However, tensile strength decreased from 0.970 MPa to 0.507 MPa (48% reduction), and elongation at break decreased from 51.00% to 44.20% (13% reduction), suggesting that frankincense reinforcement comes at the expense of ductility at this concentration. Antibacterial testing against Staphylococcus aureus, a common wound pathogen, demonstrated that the PVA/frankincense blend exhibited significant antibacterial activity, while pure PVA showed limited or no activity. This antimicrobial effect is attributed to bioactive constituents of frankincense. The PVA/frankincense electrospun nanofiber mat combines hydrophilic surface properties, enhanced stiffness, and potent antibacterial activity, making it a promising candidate for skin substitution.