This study aimed to investigate the fundamental properties, solubility, mechanical properties, barrier performance, and microstructural features of films composed of corn starch and glycerol. Changes in the microstructure were analyzed to understand how they relate to the physical and chemical properties of these films. Specifically, we found that increasing the gelatinization time decreased the film thickness, solubility, water vapor permeability, and maximum degradation temperature and increased the water content. A gradual increase in the water contact angle of the corn starch–glycerol films was observed with increasing gelatinization time. This trend is likely due to the disruptive effect of gelatinization on the crystalline and amorphous structures inherent in corn starch, resulting in reduced film crystallinity, degree of order (DO) and degree of double helix (DD).