Oleic acid and glycerol are two different molecules with different plasticization mechanisms, and their effects on the mechanical properties of zein film were investigated. The mechanical parameters of tensile strength (TS) and elongation to break (E) were improved by either oleic acid or glycerol in a dose-dependent manner up to 20%. When a combination of the two plasticizers in different ratios was used to make zein films, the highest TS was observed at a ratio of 3:1 of oleic acid to glycerol, and a synergy between them was revealed through a statistical analysis. A significant decrease of glass transition temperature (Tg) and different microscopic patterns examined by scanning electronic microscopy (SEM) indicate the supramolecular structure was changed by the plasticizers. A combination of plasticizers with different plasticization mechanisms might be a better way to synergistically improve the mechanical properties of biopolymer-based films for practical applications.