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<i>Ciona</i> molecule against microbes-A24 (CiMAM) isolated from the marine chordate <i>Ciona intestinalis</i> is an antimicrobial peptide. To generate CiMAM-expressing transgenic <i>Bacillus subtilis</i>, we constructed a plasmid expressing recombinant CiMAM (rCiMAM) and introduced it into <i>B. subtilis.</i> Transgenic strains C117 and C166 were selected since they were able to highly and stably express rCiMAM. We studied the bactericidal activity of pepsin-digested extracts from rCiMAM-expressing strains against freshwater and euryhaline pathogens that commonly occur in aquaculture ponds and found no difference from that of lactoferricin-expressing strains. The bactericidal activity of 1-μL aliquot from a total 5.5 mL extracted from 5 mL of cultured C117 (1.45 × 10⁸ CFU·mL⁻¹) and C166 (2.17 × 10⁸ CFU·mL⁻¹) against halophilic bacteria was equivalent to the efficacy of 57.06 and 32.35 ng of Tetracycline against <i>Vibrio natriegens</i>, 47.07 and 25.2 ng against <i>V. parahaemolyticus</i>, and 58.17 and 36.55 ng against <i>V. alginolyticus,</i> respectively, indicating higher bactericidal activity of pepsin-extracts from rCiMAM-containing strains against halophilic bacteria compared to that from lactoferricin-containing strains. Since the antibacterial activity of rCiMAM-expressing <i>B. subtilis</i> strains shows higher competence against halophilic pathogens compared to that against freshwater and euryhaline pathogens, these strains are promising candidates to protect marine fish and shellfish from halophilic bacterial infection.