¹Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, SAUDI ARABIA DOI : 10.9775/kvfd.2025.35575 The overuse of antibiotics in poultry farming underscores the need for safe, effective alternatives to produce clean meat. This study aimed to isolate and evaluate probiotic bacteria with strong antibacterial activity for broiler production. Thirty bacterial isolates were obtained from broiler feces samples, and three isolates were selected and identified using MALDI-TOF MS as Paenibacillus polymyxa EB7, Bacillus licheniformis EB14, and Bacillus mycoides EB26. These isolates were screened for their strong inhibitory activity against pathogens, including Staphylococcus aureus and Pseudomonas aeruginosa. Among these, P. polymyxa EB7 emerged as the most promising, combining high antibacterial and antioxidant activities with exceptional tolerance to acidic pH and bile salts. Importantly, EB7 was sensitive to major antibiotics (tetracycline, azithromycin, erythromycin, and gentamicin) and showed no hemolytic or cytotoxic activity, confirming its safety profile. In vivo broiler trials confirmed its effectiveness. Dietary supplementation with EB7 at 200 mg/kg significantly improved growth performance (body weight gain and feed conversion ratio) and mitigated the negative effects of the Salmonella challenge. EB7 supplementation improved blood biochemistry by lowering liver and kidney stress markers and oxidative damage (malondialdehyde), while increasing antioxidant enzymes (SOD, GSH, and CAT) and immune markers (IgG and IgA). It also modulated the gut microbiota by reducing pathogenic loads (E. coli, Salmonella) and increasing beneficial lactic acid bacteria. Furthermore, EB7 helped balance the immune response to Salmonella infection by modulating key immune-related genes (TLR4, IL-6, and AvBD6), reducing excessive inflammation while maintaining host defenses. The findings demonstrated that P. polymyxa EB7 is a safe, multifunctional probiotic that enhances growth, strengthens immunity, and improves gut health in broilers. Keywords : Probiotics, Human health, Gut microbiota, Salmonella challenge, Pathogenic bacteria