While brackish water (BW) irrigation can alleviate constraints on cotton industry in arid regions caused by freshwater (FW) scarcity, it also risks deteriorating soil water-salt-nitrogen conditions and reducing yield. Bacillus subtilis (B. subtilis) has demonstrated potential for mitigating such adverse effects, yet its specific efficacy and regulatory mechanisms remain insufficiently understood. Thus, in the arid region of Xinjiang, China, we applied 0 and 45 kg & sdot;ha-1 of B. subtilis to soil under FW and BW irrigation to investigate the regulatory effect of B. subtilis on soil conditions and cotton (Tahe No. 2) production. Results showed that B. subtilis inhibited soil salinization induced by BW irrigation and mitigated water use impairment and nitrogen loss. Improved soil conditions increased cotton leaf chlorophyll content, net photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (Gs), while reducing intercellular CO2 concentration (Ci). Moreover, applying B. subtilis improved instantaneous water use efficiency (iWUE), intrinsic water use efficiency (IWUE), and instantaneous carboxylation efficiency (CE). These improvements in photosynthetic traits further promoted cotton growth and development, ultimately ensuring its water and nitrogen use efficiency and production security. Furthermore, B. subtilis improved soil microbial community alpha diversity and composition, enhanced the synergy and modularity of species interactions, and thereby strengthened metabolic functions associated with salt stress regulation and nitrogen transformation. The optimization of bacterial community alpha diversity and functions significantly improved soil water-salt-nitrogen conditions and cotton production under both FW and BW irrigation. Notably, fungal community played an insignificant role in driving the comprehensive improvement under FW irrigation. However, under BW irrigation, B. subtilis effectively modulated the interactive balance between fungal and bacterial communities. This regulation significantly enhanced the contribution of fungal community to the improvement effect, consequently achieving a more efficient promotion of cotton production. Overall, our results confirmed that B. subtilis can effectively enhance BW irrigation safety and elucidated the important mechanisms of action therein, showcasing feasible microbial regulation technology for alleviating FW scarcity constraints on cotton industry in arid regions.