Four weeks feeding trial was conducted to assess the in vivo probiotic potentials of three host-associated Bacillus species in O. niloticus. The fish were divided into five treatment groups and fed with diets containing no bacteria (group CT), B. velezensis TPS3N (group V), B. subtilis TPS4 (group S), B. amyloliquefaciens TPS17 (group A) and a 5th group which comprised the combination of the three Bacillus species at a ratio 1:1:1 (group CB). At the end of the feeding trial, enhanced growth performance in terms of final weight (FW), weight gain (WG), specific growth rate (SGR) as well as significantly lower (P < .05) feed conversion ratio (FCR) were recorded in the Bacillus treatment groups with the CB group having the best performance. Improved immune and biochemical (P < .05) (nitric oxide (NO), immunoglobulin M (IgM), lysozyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (AKP)) indexes, antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) and digestive enzymes (lipase and trypsin) were also recorded in the treatment groups relative to the CT group. Significantly lower (P < .05) GOT and MDA activities were also observed in the V, S, A, and CB groups than the CT groups. Growth-related genes GHR-1 and IGF-1 were upregulated significantly (P < .05) in both liver and head kidney (HK) of fish fed Bacillus supplemented diets. Likewise, the CB group induced significant upregulation (P < .05) of TNF-α, TLR-2, IgM (except in the liver) and C-LYZ in both liver and HK. A subsequent challenge test with S. agalactiae resulted in cumulative mortalities (%) of 60%, 33%, 27%, 20%, and 13% in the CT, V, S, A, and CB treatment groups respectively. It was therefore concluded from this experiment that dietary inclusion of the Bacillus species singularly or in combination could elicit probiotic effects and protect O. niloticus against S. agalactiae infection.