A mixed natural preservative composed of ε-polylysine (ε-PL), chestnut 70% ethanol extract (NE), and cinnamon hydrothermal extract (CW), was investigated for the reduction of Staphylococcus aureus. The minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) of seven natural extracts were investigated against a cocktail of three strains of S. aureus (ATCC 25923, ATCC 33591, and ATCC 33594). Three important factors (ε-PL, NE, and CW) were selected by using the Plackett-Burman (PB) design for the response surface model (P < 0.001). Following a central composite design, S. aureus were treated with mixtures of natural preservatives that included ε-PL, NE, and CW. The MIC and MBC of ε-PL and the natural extracts and ranged from 1 to 16 mg/mL (R2 = 0.9857). The mixed natural preservative presented a synergistic antibacterial effect, at the optimum point. These results suggest that mixed natural preservatives of ε-PL, NE, and CW can lower the economic cost of food processing.