Antagonistic activities against candidate indicator strains, adhesion to mucus and biofilm formation of potential probiotic strains isolated from Poecilia reticulata were evaluated. Four isolated strains (MBTU_PB1, MBTU_PB2, MBTU_PB3 and MBTU_PB4) showed moderate to strong antagonistic activities against the tested five indicator strains (Aeromonas hydrophila1739, Vibrio cholera 3906, Flavobacterium 2495, Acinetobacter 1271 and Alcaligenes 1424) and these isolates were further identified using biochemical tests and 16S rDNA gene sequence analysis. Except the whole cell product, the other three cellular components, namely, heat-killed whole cell product, intracellular product and extracellular product of all the four selected isolates were equally effective, as revealed by the zone of inhibitions to the tested indicator strains. The in vitro adhesion property or the ability of colonization is often considered as a selection criteria for probiotics. All the selected four strains had higher adhesion abilities than the indicator strains. Further, these four strains had the ability to form biofilms on polystyrene surfaces. The in vitro characterization of these four strains suggests possibility of using the isolates, as individual strain or in combination, for probiotic therapy in aquaculture.