Abstract: Chemical preservatives are used to control spoilage microorganisms in food. Preservative residues have certain potential hazards to the human body. In this paper, lactic acid bacteria resistant to food spoilage fungi were screened and compounded and mixed fermentation were carried out to improve the antifungal activity of the fermentation broth. The 24-well double-layer agar method and the graded inhibitory concentration method were used to screen the more antifungal strains from multiple strains of lactic acid bacteria and Propionibacterium and determine the optimal combination of mixed bacteria. Through the Plackett-Burman test and the steepest climbing test, the fermentation process was optimized, and the antifungal activity of the fermentation supernatant was measured with a 96-well plate microplate reader method. The results showed that the optimal combination of mixed bacteria was Lactobacillus plantarum L9 and Propionibacterium frederi D5. The optimized formula of fermentation medium: Glucose 55.0 g/L, calcium carbonate 6.7 g/L, yeast extract 14.8 g/L, hydrogen phosphate dipotassium 0.25 g/L, manganese sulfate 0.1 g/L, sodium acetate 5.0 g/L, ammonium citrate 2.0 g/L, the inoculation ratio was 5:1 (D5:L9), and the fermentation temperature was 37 °C. The optimized fermentation medium of the selected L9 and D5 was verified by mixed fermentation, and the antifungal activity of the fermentation broth could be as high as 47.07AU.