Skin and intestinal mucosa lymphoid tissues are known to be the fish's first line of defence since they serve as the first point of contact for pathogens. Only few studies have investigated the influence of host-associated Bacillus on mucosal immunity. In this study, the effects of three host-associated Bacillus species on mucosal immunity, intestinal morphology, intestinal digestive enzymes activity, intestinal microbiome and resistance of Nile tilapia against Aeromonas hydrophila infection was evaluated. The fish were divided into five treatment groups and fed with diets containing no bacteria denoted as Control, Bacillus velezensis TPS3N denoted as group V, Bacillus subtilis TPS4 denoted as group S, Bacillus amyloliquefaciens TPS17 denoted as group A and a 5th group containing the three Bacillus species at a ratio 1:1:1 denoted as group CB. At the end of the feeding trial, significant enhancement of both skin mucus and intestinal immune titres were recorded in terms of nitric oxide (NO) (except in the mucus of V and S groups), immunoglobulin M (IgM) (except in the intestine of group V), lysozyme (LZM), and alkaline phosphatase (AKP) in all fish fed the Bacillus supplemented groups relative to the untreated group. Intestinal antioxidant enzymes (catalase (CAT) (except in the intestine of group S) and superoxide dismutase (SOD)) capacity of Nile tilapia were higher in the Bacillus groups. Intestinal lipase activity was elevated in the Bacillus supplemented groups. The intestinal morphological parameters (villus height, villus width, goblet cells count (except in group S and A), and intestinal muscle thickness) were significantly enhanced in the Bacillus supplemented groups relative to the Control group. Dietary probiotic supplementation also influenced the intestinal microflora composition of Nile tilapia. Proteobacteria recorded the highest abundance followed by Firmicutes, Fusobacteria, and Bacteroidetes at the phylum level in this study. At the genus level, the abundance of pathogenic bacteria viz Staphylococcus and Aeromonas were reduced in the Bacillus supplemented groups in comparison to the Control group. A challenge test with A. hydrophila resulted in lower mortalities (%) in the Bacillus treated groups thus 86.67%, 50.00%, 43.33%, 63.33%, and 30.00% for Nile tilapia fed Control, V, S, A, and CB diets respectively. In conclusion, the inclusion of B. velezensis TPS3N, B. subtilis TPS4, and B. amyloliquefaciens TPS17 in the diet of Nile tilapia singularly or in combination, could enhance the mucosal immunity, intestinal health, and resistance of Nile tilapia against A. hydrophila infection.