The fermentation medium of Micromonospora Y15 was optimized to enhance the production of antimicrobial metabolites with the antimicrobial activity tested against Listeria monocytogenes. First, the optimal medium components (carbon source, nitrogen source and inorganic salt) were determined by the single factor experiment. Second, the Plackett–Burman experiment was used to screen significant factors. Third, the steepest ascent experiment was undertaken to figure out the optimal region of the significant factors. Finally, the central composite experiment was conducted to optimize the final medium components. The single factor experiment results suggested that the optimal medium components were soluble starch, beef extract, peptone, NaCl, K₂HPO₄, MgSO₄, and FeSO₄, while the Plackett–Burman experiment demonstrated that beef extract, K₂HPO₄ and soluble starch were significant factors affecting antimicrobial activity. The steepest ascent experiment results further showed the central point of three significant factors were 1.00 g/L of K₂HPO₄, 25.00 g/L of soluble starch and 18.00 g/L of beef extract, respectively. Response surface analysis revealed that the optimum values of the tested significant variables for the production of antimicrobial metabolites were 1.03 g/L of K₂HPO₄, 27.67 g/L of soluble starch and 18.30 g/L of beef extract, accordingly. Under this optimal condition, the antimicrobial activity of the fermented medium was 1236.79 ± 14.56 AU/mL, which increased 3.86 times compared with the initial medium. We thus concluded that the medium composition optimized in the study would be helpful for the production of antimicrobial metabolites by Micromonospora Y15.