Bacterial illness causes detrimental impacts on fish health and survival and finally economic losses for the aquaculture industry. Antibiotic medication causes microbial resistance, so alternative control strategies should be applied. In this work, we investigated the probiotic-medicated diet as an alternative control approach for antibiotics in treating Vibrio cholerae infection in Nile tilapia (Oreochromis niloticus). One hundred eighty fish (50 ± 2.5 g Mean ± SD) were allocated into six groups in glass aquariums (96 L) in triplicate for 10 days. Groups 1 (G1), G2, and G 3 were intraperitoneally (IP) injected with 0.5 mL sterilized tryptic soy broth and fed on a basal diet, basal diet contained B. subtilis (BS) (1 × 10 5CFU/ kg-1 diet), and basal diet contained trimethoprim-sulfamethoxazole (TMP-SMX) (1.5 g/kg-1 diet), respectively. Additionally, G4, G5, and G6 were IP challenged with 0.5 mL of V. cholerae (1.5 × 107 CFU) and received the same feeding regime as G 1 to 3, respectively. The results exhibited that the V. cholera-infected fish exhibited skin hemorrhage, f in rot, and the lowest survival (63.33%). Additionally, lowered immune-antioxidant biomarkers (white blood cells count, serum bactericidal activity, phagocytic activity, phagocytic index, and lysozymes) with higher lipid peroxidation marker (malondialdehyde) were consequences of V. cholerae infection. Noteworthy, fish-fed therapeutic diets fortified with BS and TMP-SMX showed a substantial amelioration in the clinical signs and survival. The BS diet significantly improved (P < 0.05) the immune-antioxidant indices of the infected fish compared to the TMP-SMX diet. The current findings sup ported the use of a BS-enriched diet as an eco-friendly approach for the control of V. cholerae in O. niloticus.