Inhibiting the growth of spoilage bacteria, such as Pseudomonas spp., is key to reducing spoilage in fish. The mucus adhesion test in vitro showed that the adhesion ability of Bacillus subtilis was positively correlated with its inhibition ability to Pseudomonas spp. In vivo experiments of tilapia showed that dietary supplementation with B. subtilis could reduce the adhesion and colonization of Pseudomonas spp. in fish intestines and flesh, as well as reduce total volatile basic nitrogen (TVB-N) production. High throughput and metabolomic analysis showed treatment with B. subtilis, especially C6, reduced the growth of Pseudomonas spp., Aeromonas spp., Fusobacterium spp., and Enterobacterium spp., as well as aromatic spoilage compounds associated with these bacteria, such as indole, 2,4-bis(1,1-dimethylethyl)-phenol, 3-methyl-1-butanol, phenol, and 1-octen-3-ol. Our work showed that B. subtilis could improve the flavor of fish by changing the intestinal flora of fish, and it shows great promise as a microecological preservative.