Bacillus subtilis, a probiont isolated from the intestine of Cirrhinus mrigala (Hamilton), was incorporated in fish feed at four different concentrations (5 × 108, 5 × 107, 5 × 106 and 5 × 105 cells g−1) and fed to Poecilia reticulata (Peters), Poecilia sphenops (Valenciennes), Xiphophorus helleri (Heckel) and Xiphophorus maculatus (Gunther) for a period of 90 days. The growth parameters, proximate composition of whole body, digestive enzymes and gut microbiology were monitored at regular monthly intervals. The increments in length and weight and the survival were significantly higher (P < 0.05) and the values of food conversions were significantly lower (P < 0.05) in fishes fed the probiotic feeds. The administration of B. subtilis in diet resulted in an increase (P > 0.05) in body ash and protein content and in the specific activity of protease and amylase in the digestive tract of all the fishes. The counts of total heterotrophs, motile aeromonads, presumptive pseudomonads and total coliforms were significantly lower (P < 0.05) in the gut of the probiotic fed fishes. Bacillus subtilis persisted in the fish intestine and in the feed in high numbers during the feeding period. The high survival and low infectivity recorded in the probiotic feed fed fishes when challenged by immersion assay with the pathogenic Aeromonas hydrophila demonstrate that the probiotic strain confers disease resistance in live-bearing ornamental fishes. A probiotic concentration of 106 to 108 cells g−1 is adequate for improved growth performance and survival and for healthy gut microenvironment and use of higher concentration of the probiont did not always lead to significantly better results.