Abstract Chitosan (CS) with excellent biomedical properties was mixed with polyvinyl alcohol (PVA) to be used as the spinning solution. The spinning solutions with various concentrations of CS:PVA from 10:90% to 50:50% (v/v) were investigated. Tween 80 (T80) was added in the spinning solutions of CS and PVA. The nanofiber mats with and without T80 addition obtained from the spinning solutions by electrospinning technique were investigated and addressed. The results showed that the viscosity of the CS and PVA spinning solutions increased with increasing the CS concentration, whereas the viscosity decreased after T80 addition. The nanofiber mats with 10–30% CS concentrations were prepared successfully as a smooth surface and high dense nanofiber mat. The average diameter of the nanofiber decreased with increasing the CS concentration. The increase in the CS concentration of the nanofiber mat can increase the mechanical and antibacterial properties, whereas the wettability and drug release property were decreased. Moreover, the nanofiber mats with T80 addition had higher mechanical property and wettability than the nanofiber mats without T80 addition. Finally, the T80 addition can enhance hydrophilicity and promote the drug release property of the nanofiber mat.