The beneficial effects of two probiotic bacterial strains Marinilactibacillus piezotolerans and Novosphingobium sp. during the culture of Indian white shrimp, Penaeus indicus, under biofloc and clear water system were evaluated. The experimental variation were CW1 (M. piezotolerans in clear water), BFT1 (biofloc + M. piezotolerans), CW2 (Novosphingobium sp. in clear water), BFT2 (biofloc + Novosphingobium sp.) and control (without bacterial strains and biofloc). Growth and survival considerably increased in probiotic bio-augmented treatments. Probiotic incorporation significantly improved water quality, especially ammonia reduction. Microbiota analysis from gut samples taken from different treatments revealed varied microbial population structure among clear water culture, biofloc culture and control. Proteobacteria and Firmicutes were the top phyla observed in the treatments which were significantly higher in bio-augmented systems than the control. Vibrio genera were predominantly observed in control and clear water system compared to that of biofloc systems. Immune genes were significantly altered in response to probiotic gut microbial supplementation than the control. Higher gene expression profile of important immune genes was observed in the biofloc reared shrimps. Expression of digestive enzyme related genes such as trypsin, chymotrypsin, cathepsin L, cathepsin B and alpha amylase were also upregulated significantly in probiotic supplementation especially in the biofloc treatments. Proteomic analysis of hepatopancreas of shrimps from different treatments was carried out by using 2D gel electrophoresis and MALDI-TOF analysis. The proteins were mostly related to growth and stress tolerance. Eukaryotic initiation factor 4E binding protein was expressed in all the groups and it was high in biofloc treated animals followed by animals treated solely with probiotics compared to those of control groups. The results concludes that biofloc already proved as an effective culture method for healthy shrimp production and supplementation of probiotic bacterial strains registered additional benefit for growth, survival, microbial, immunological status of P, indicus culture.