In this study, polyethylene oxide (PEO) and curdlan solutions were used to prepare PEO/curdlan nanofiber films by electrospinning using deionized water as the solvent. In the electrospinning process, PEO was used as the base material, and its concentration was fixed at 6.0 wt.%. Moreover, the concentration of curdlan gum varied from 1.0 to 5.0 wt.%. For the electrospinning conditions, various operating voltages (12–24 kV), working distances (12–20 cm) and feeding rates of polymer solution (5–50 μL/min) were also modified. Based on the experimental results, the optimum concentration for the curdlan gum was 2.0 wt.%. Additionally, the most suitable operating voltage, working distance and feeding rate for the electrospinning process were 19 kV, 20 cm and 9 μL/min, respectively, which can help to prepare relatively thinner PEO/curdlan nanofibers with higher mesh porosity and without the formation of beaded nanofibers. Finally, the PEO/curdlan nanofiber instant films containing 5.0 wt.% quercetin inclusion complex were used to perform wetting and disintegration processes. It was found that the instant film can be dissolved significantly on the low-moisture wet wipe. On the other hand, when the instant film touched water, it can be disintegrated very quickly within 5 s, and the quercetin inclusion complex was dissolved in water efficiently. Furthermore, when the instant film encountered the water vapor at 50 °C, it almost completely disintegrated after immersion for 30 min. The results indicate that the electrospun PEO/curdlan nanofiber film is highly feasible for biomedical applications consisting of instant masks and quick-release wound dressings, even in the water vapor environment.