Probiotics, live cells with different beneficiary characteristics, have been extensively studied and explored commercially in many different products in the world. This study was aimed to isolate potential probiotic bacterial isolates from different sources and to study its inhibitory properties against fish pathogenic bacteria. Isolation of bacteria from curd and milk samples and diseased fishes were conducted on MRS agar, nutrient agar and BHI agar respectively. The identifications were carried out using, morphological and biochemical and molecular methods. The two bacterial isolates obtained were then checked for antagonism within them and against the fish pathogen. Optimization studies for pH, temperature, carbon and nitrogen sources were also conducted for these two probiotic strains. The isolates obtained from curd, milk and diseased fishes were morphologically and biochemically identified as Bacillus subtilis, Lactobacillus acidophilus and Vibrio alginolyticus respectively. Bacillus subtilis and Lactobacillus acidophilus showed no antagonism against each other whereas their bacterial consortium found to have excellent antagonism against Vibrio alginolyticus. Optimization of various parameters found to be associated with the increased production of Bacillus subtilis and Lactobacillus acidophilus. Probiotic properties were assayed including acid tolerance, pH tolerance, temperature tolerance and estimation of lactic acid produced. Thus the isolated bacteria can be further used in the development of probiotic therapy for fishes.