In this study, the gluten nanofibrous films incorporated with glycerol monolaurate (GML) were fabricated through electrospinning. With the increasing concentration of GML, the surface tension of the gluten solution decreased continuously, while the viscosity decreased at a lower concentration of 4% GML but then increased significantly. Compared to the spindle-in-string fiber morphology of the electmspun gluten film, the addition of GML resulted in uniform smooth nanofibers with diameters ranging from 317 to 149 nm. As indicated by the XRD and FTIR analysis, GML was uniformly dispersed in the gluten films through hydrogen bonding at lower concentrations, but crystalized at higher concentrations, leading to a stiffer but brittle network. The incorporation of GML caused a more hydrophilic film surface as evidenced by the more accelerated decrease in water contact angle, but endowed the gluten nanofibrous films with a superior water stability and excellent antimicrobial activities against Staphylococcus aureus and Escherichia coli. These results suggested the potentials of the electrospun GML incorporated gluten films with superior water stability for antimicrobial food-packaging applications.