The ethylcellulose/gelatin solutions containing various concentrations of zinc oxide (ZnO) nanoparticles were electrospun, and the resultant nanofibers were characterized by scanning electron microscopy, energy dispersive X-ray, X-ray photoelectron spectrometer, X-ray diffraction, Fourier transform infrared spectroscopy, mechanical testing, water contact angle, and water stability. Results indicated that ZnO nanoparticles acting as fillers interacted with polymers, resulting in the enhanced surface hydrophobicity and water stability of nanofibers. The antibacterial assay showed a concentration-dependent effect of ZnO on the viabilities of Escherichia coli and Staphylococcus aureus. Notably, the antimicrobial efficiency of the 1.5 wt % ZnO containing fibers against Staphylococcus aureus was 43.7% but increased to 62.5% after UV irradiation at 364 nm, possibly due to the significantly increased amounts of intracellular reactive oxygen species. These results suggested that the ZnO-containing nanofibers with excellent surface hydrophobicity, water stability, and antimicrobial activity exhibited potential uses in food packaging.