Comparison of the growth performance of Penaeus vannamei was ascertained by supplementing the probiotics from commercial and biofloc system incorporated through feed. Post larvae of shrimp (0.045 ± 0.005g) were stocked at a density of 500/m3 in FRP tanks (500L) in triplicates for a period of 60days. A total of 40 bacterial strains were isolated from previous biofloc culture trials were tested for its antimicrobial activity against the pathogen Vibrio parahaemolyticus. Out of which, Klebsiella pneumoniae, Bacillus megaterium, Exiguobacterium profundum, Pseudomonas balearica and Pseudomonas stutzeri showed higher antimicrobial activity. The treatment group includes Biofloc (C), commercial probiotic (T1), isolated probiotic (T2), Biofloc + commercial probiotic (T3) and Biofloc + isolated probiotic (T4) in triplicates. Distellary spentwash was used as a carbon source for biofloc development and maintenance. The probiotic concentration of 1x109 cfu/g were supplemented throughout the trial. The recorded water quality parameters (pH, alkalinity, Calcium and Magnesium) were observed to be significant among the experimental groups (P≤ 0.05). Highest weight gain (2.43g), SGR, PER and lower FCR values were recorded in T4. Lowest values of total Vibrio was found in T3. Histology analysis revealed that there was a mild increase in the B and R cell vacuoles in hepatopancreas of T1 and T4 whereas mild degeneration was found in intestine of T1 and T2.Microbiome analysis of the shrimp gut revealed that Proteobacteria was found to be the most abundant phylum in all the experimental groups. P. balearica, K.pneumoniae,P. stutzeri and E.profundum were found to be present in the gut of C whereas P. balearica, K. pneumonia and P.stutzeri in the gut of T2 and T4.The results proved that the probiotics isolated from biofloc colonized in shrimp gut would pave a promising role in aquaculture.