Listeria monocytogenes is an important foodborne pathogen, and the development of safe and efficient natural antibacterial agents is of great significance.The objective of this study was to evaluate the safety of the bacteriocin-producing Enterococcus mundtii A9-1A strain targeting L.monocytogenes and to optimize its fermentation conditions for enhanced bacteriocin yield. The in vitro and in vivo animal experiments were conducted in this study. The results showed that no hemolytic activity or antibiotics resistance was observed. It had no significant adverse effects on the health of normal mice in terms of body weight, organ indices, serum liver function indicators, cytokines, and organ tissues, with no bacterial translocation observed. Through single-factor tests and uniform design, the optimal medium composition was determined as follows: yeast extract 20.00 g·L-1, glucose 20.00 g·L-1, Tween 80 2.74 mL·L-1, K2HPO4·3H2O 4.41 g·L-1, CH3COONa·3H2O 3.24 g·L-1, ammonium citrate 9.91 g·L-1, MgSO4·7H2O 0.60 g·L-1, and MnSO4·4H2O 1.00 g·L-1. After medium optimization, the diameter of the inhibition zone reached 42.82 mm, representing an increase of 10.73% compared with the initial medium (38.67 mm). The optimal fermentation conditions were determined by using orthogonal experiments, which were listed as follows: culture time 24 h, temperature 30 ℃, initial pH 7.0, and inoculation ratio 3.0% (φ). Following the combined optimized medium composition and culture conditions, the inhibition zone diameter increased to 45.09 mm, which was 16.60% higher than that of the initial medium. Meanwhile, the bacteriocin titer increased from 6826.67 AU·mL-1 to 10240.00 AU·mL-1, with an increase rate of 50.00%. These results provide a solid data foundation for improving the bacteriocin production of E.mundtii and its application as a biological agent.