Abstract: Vibrio parahaemolyticus is a highly pathogenic bacterium in aquaculture, and the overuse of antibiotics has led to increasingly severe issues of antimicrobial resistance and ecological risks. We optimized the fermentation process of a marine-derived Bacillus atrophaeus strain N1 to enhance the production of its antibacterial substances (Antibacterial substances from N1, AsN). Besides, we evaluated the protective efficacy of AsN against V. parahaemolyticus infection by using a Manila clam (Ruditapes philippinarum) infection model, so as to provide a novel strategy for the eco-friendly control of V. parahaemolyticus in aquaculture. We systematically optimized the fermentation conditions (Temperature, pH, inoculum size, time) and medium components (Nitrogen source, carbon source, NaCl) through single-factor experiments combined with Plackett-Burman design, steepest ascent experiments and Box-Behnken response surface methodology. We also purified the antibacterial substance AsN using DEAE anion-exchange chromatography followed by Sephadex G-100 gel filtration chromatography, yielding an antimicrobial protein with an approximate molecular mass of 63 kDa. Comprehensive characterization included evaluation of temperature/pH stability, salt tolerance and metal ion effects. Finally, a R. philippinarum infection model was established to validate both the inhibitory effects of AsN against V. parahaemolyticus and its survival rate enhancement capability. The optimized fermentation process enhanced AsN production by 16.58% (Inhibition zone diameter: 17.16 mm), with optimal conditions identified as 37 ℃, pH 8.0, 6% (φ) inoculum and 30 h cultivation. The refined medium composition comprised 10 g·L⁻¹ soy protein isolate, 11 g·L⁻¹ yeast extract, and 12 g·L⁻¹ NaCl. Purification yielded a 63 kD antimicrobial protein exhibiting peak activity at 4°C and pH 7.0, while maintaining efficacy across broad ranges (Temperature: 4–100 ℃; pH: 2.0–9.0). The protein demonstrated exceptional salt tolerance, retaining high antibacterial activity after 24 h in saturated NaCl solution. Ferric ions (Fe³⁺) potentiated its inhibitory effects, whereas barium (Ba²⁺), manganese (Mn²⁺), and calcium (Ca²⁺) ions suppressed activity. In vivo trials confirmed AsN significantly improved clam survival rates (80%, comparable to antibiotics, p<0.05). These findings position AsN as a thermally stable, salt-resistant alternative to antibiotics for combating V. parahaemolyticus infections.