Melanoidins produced from the combination of D-xylose and L-phenylalanine have been reported to exhibit strong antibacterial effects. This study investigated the influence of environmental factors, such as temperatures (10, 15, 20, 25, 30, 35, 40, and 45°C), pH (5.5, 6.0, 6.5, 7.0, 7.5, and 8.0), and water activity (aw: 0.99, 0.96, and 0.93), on the antibacterial effect of the melanoidins produced from the combination of D-xylose with L-phenylalanine against Bacillus cereus and Clostridium perfringens in culture media. Furthermore, freeze-dried powdered melanoidin was used to determine the minimum concentration for growth inhibition, to compare the antibacterial effect of the melanoidin with conventional food preservatives. The liquid melanoidins significantly inhibited the growth of B. cereus (up to 4 log CFU/mL at the maximum) and C. perfringens (up to 6.5 log CFU/mL at the maximum) regardless of the incubation temperatures. However, the remarkable difference between the presence and absence of the melanoidins was demonstrated in the range of 20–35°C as 4 log-cycle lower in B. cereus and 2 log-cycle lower in C. perfringens than those without the melanoidins. The antibacterial effect of the melanoidin on B. cereus was not influenced by pH from 5.5 to 7.0, which exhibited 2–3 log-cycle lower viable counts than those without the melanoidin. Only one log-cycle difference between with and without the melanoidin was shown in C. perfringens growth under the pH range of 5.5–7.0. Although there was no significant difference in the growth of B. cereus between three aw conditions, the melanoidin exhibited a significant antibacterial effect at aw 0.99 demonstrating 4 log-cycle lower viable numbers than those without the melanoidin. Minimum inhibitory concentration of the melanoidin powder for B. cereus and C. perfringens was 7 mg/mL and 15 mg/mL, respectively, regardless of the kind of foods. Furthermore, the melanoidin exhibited comparable antibacterial effect on B. cereus and C. perfringens to potassium sorbate and sodium benzoate under the same concentration as the minimum inhibitory concentration of the melanoidin, demonstrating 2 log-cycle reduction during 3 days of incubation period at 25°C. The results presented here suggest that the xylose- and phenylalanine-based melanoidin demonstrates the possibility to be an alternative food preservative.