Abstract: F34-F44 is a compatible and highly antagonistic combination of two Bacillus velezensis strains screened and constructed from antagonistic bacteria against Erwinia amylovora, the causal agent of fire blight, and has potential for development as a biopesticide. To improve the fermentation performance and biocontrol efficacy of F34-F44, this study employed viable cell count and antibacterial activity as the main response indicators, supplemented by endospore count and sporulation rate. The medium composition and shake-flask fermentation conditions were systematically optimized through single-factor, Plackett-Burman, and response surface methodology. The optimized medium for F34-F44 consisted of 27.6 g/L molasses, 7.5 g/L cottonseed maceration powder, and 1 g/L KH₂PO₄. The optimal fermentation conditions were as follows: temperature 31.8 ℃, rotation speed 222 r/min, initial pH 7.0, inoculum volume 3%, and fermentation duration of 48 h. After optimization, the count of viable bacteria, endospore count, sporulation rate, and inhibition zone diameter of F34-F44 fermentation broth reached 3.22×10⁹ CFU/mL, 2.66×10⁹ CFU/mL, 82.61% and 64.9 mm, respectively, representing 6.19-, 10.64-, 1.72-, and 1.73-fold improvements compared with pre-optimization levels. The antibacterial activity of the combined bacteria fermentation broth remained above 88% and the viable cell count of F34-F44 fermentation broth remained at 10⁹ CFU/mL after treatment with 100 ℃ for 30 min or UV exposure for 1 h. The fermentation broth exhibited relatively stable antibacterial activity and high viable cell counts within the pH range of 7.0-9.0, but was sensitive to extreme acid or alkali conditions. The fermentation broth also exhibited a certain tolerance to trypsin, pepsin, and proteinase K. The results showed that the F34-F44 fermentation broth exhibited strong and stable antibacterial activity and stability. The protective efficacy of the F34-F44 fermentation broth against fire blight was evaluated using detached inflorescences of Pyrus sinkiangensis. The results showed that the spraying optimized F34-F44 fermentation broth significantly reduced blossom blight incidence (P < 0.05), achieving a protective efficacy of 76.70% against fire blight, which was 9.97% higher than that obtained with the pre-optimization fermentation. These results provide a basis for the development of the F34-F44 combined microbial agent and its application in the biological control of fire blight.