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The adoption of intensive farming has exacerbated disease outbreaks in aquaculture, particularly vibriosis caused by <i>Vibrio parahaemolyticus</i>. The use of probiotics to control <i>V. parahaemolyticus</i> is recognized as a good alternative to antibiotics for avoiding the development of antibiotic-resistant bacteria. In this study, two strains of B. HLJ1 and B. C1 with strong inhibitory activity on <i>V. parahaemolyticus</i> were isolated from aquaculture water and identified as <i>Bacillus subtilis</i> and <i>Bacillus pumilus</i>, respectively. Both B. HLJ1 and B. C1 lacked antibiotic resistance and virulence genes, suggesting that they are safe for use in aquaculture. In addition, these two strains can tolerate acid environments, produce spores, secrete extracellular enzymes, and co-aggregate as well as auto-aggregate with <i>V. parahaemolyticus</i>. B. HLJ1 and B. C1 produced the same anti-<i>V. parahaemolyticus</i> substance, which was identified as AI-77-F and belongs to amicoumacins. Both B. C1 and B. HLJ1 showed inhibitory activity against 11 different <i>V. parahaemolyticus</i> and could effectively control the growth of <i>V. parahaemolyticus</i> in simulated aquaculture wastewater when the concentration of B. C1 and B. HLJ1 reached 1 × 10⁷ CFU/mL. This study shows that B. HLJ1 and B. C1 have great potential as aquaculture probiotics.