In recent years, there has been a growing interest in the use of probiotics in aquaculture, due to their effectiveness on production, safety, and environmental friendliness. Probiotics, used as feed additives and as an alternative to antibiotics for disease prevention, have been shown to be active as growth promoters, improving survival and health of farmed fish. In this study, we have investigated the ability of the strain Bacillus velezensis MT9, as potential probiotic, to modulate the intestinal microbiota of the Nile tilapia (Oreochromis niloticus) fed with the Bacillus velezensis-supplemented feed in an experimental aquaculture plant. The analysis of the microbial community of the Nile tilapia by culture-based and 16S rRNA gene metabarcoding approaches demonstrated that B. velezensis MT9 reshapes the fish intestinal microbiota by reducing the amounts of opportunistic Gram-negative bacterial pathogens belonging to the phylum of Proteobacterium (Pseudomonadota) and increasing the amounts of beneficial bacteria belonging to the phyla Firmicutes (Bacillota) and Actinobacteria (Actinomycetota). Specifically, dietary supplementation of Nile tilapia with B. velezensis MT9 resulted in an increase in the relative abundance of bacteria of the genus Romboutsia, which has a well-documented probiotic activity, and a decrease in the relative abundance of Gammaproteobacteria of the genera Aeromonas and Vibrio, which include opportunistic pathogens for fish, and Escherichia/Shigella, which may pose a risk to consumers. The whole genome sequence of B. velezensis MT9 was then determined. Genome analysis revealed several peculiarities of B. velezensis MT9 compared to other B. velezensis reference strains including specific metabolic traits, differences in two-component and quorum sensing systems as well as the potential ability to produce a distinct array of secondary metabolites, which could explain the strong ability of this strain to modulate the intestinal microbiota of the Nile tilapia.