Intensification in aquaculture has led to the development of disease conditions impeding both economic and social development. Bacteria being opportunistic pathogens can easily infect fish and cause severe losses. Application of antibiotics by farmers can be environmentally hazardous by obliteration of the beneficial bacteria and potentially deleterious by development of antimicrobial resistance in the natural flora. The current study therefore was undertaken to isolate probiotic bacterial strains for the protection of cultured tilapia (GIFT) for an economically viable and sustainable aquaculture. Tilapia is the second most cultured fish around the world and genetically improved farmed tilapia is the fastest growing strain, which can survive in wide environmental conditions. Live tilapia (GIFT), water and soil samples were transported from Thanjavur, Madurai, Alapuzha and Krishnagiri districts of South India. Gut bacteria were isolated from the fish and plated in tryptone soya agar plate after sacrificing the fish. About 269 bacteria were isolated from the gut of 30 fishes and 79 isolates from soil and water of culture ponds. The isolates were screened for antagonistic activity against Aeromonas hydrophila and Streptococcus agalactiae by cross streak method. Out of 348 bacterial isolates, two were selected for their potent antibacterial activity against the two pathogens. The two probiotic bacterial strains were confirmed to be Bacillus sp. by 16srRNA gene sequencing. Further studies are underway to explore their probiotic effects.