Abstract <p>The study was intended to develop a method to enhance antagonistic activity of <i>Bacillus subtilis</i> strains by supplementing the nutrient medium for submerged cultivation with chitosan salicylate (CS) in order to improve the efficiency of multifunctional biopreparations based on these strains. The effect of different CS concentrations (0.05, 0.1, 0.2, and 0.5%) on the growth, development, and antagonistic activity of the studied strains has been evaluated. The tested <i>B. subtilis</i> strains exhibit high antagonistic activity against&#xa0;<i>Bipolaris sorokiniana</i> providing a fungal growth inhibition zone of 41 mm in diameter and effectively suppressing conidial germination (by up to 52.0%) compared to the control (87.1%). The optimal CS concentrations for medium supplementation (0.05 and 0.1%) increase the baseline antagonistic activity by ~20%, that is confirmed by the expansion of the zone of inhibition (ZOI) from 41 to 45-47 mm while maintaining conidial inhibition efficacy by 50.0 and 45.0%, respectively. The further CS concentration increase to 0.2 and 0.5% results in more than a twofold reduction of the ZOI diameter down to 17.5-17.7 mm; in addition, <i>B. subtilis</i> strains almost completely lose their ability to inhibit conidial germination in <i>B. sorokiniana</i>. This effect is likely associated with the direct negative effect of these CS concentrations on the bacterial growth, as evidenced by a tenfold decrease in bacterial cell titers (from 1.8-2.2 × 10¹¹ to 2.2-3.3 × 10¹⁰ CFU/mL.</p>