Finding effective alternatives to antibiotics is crucial for sustainable aquaculture. Host-derived probiotics have great potential as a promising alternative to antibiotics for immune regulation and disease control in fish farming. However, limited research exists regarding the application of native probiotics in largemouth bass (Micropterus salmoides). This study aims to evaluate the potential of the endogenous strain Pediococcus acidilactici B49 as a probiotic in modulating host immunity and disease control through in vitro and in vivo experiments. The results demonstrated that P. acidilactici B49 exhibited no hemolytic activity and displayed susceptibility to most tested antibiotics. It successfully survived and colonized in the intestinal tract of the largemouth bass. Furthermore, this strain showed remarkable antibacterial activity against common aquatic pathogens, including gram-positive and gram-negative bacteria, and also exhibited resistance against Aeromonas hydrophila on the head kidney leukocytes of largemouth bass in vitro. Following an 8-week feeding trial, P. acidilactici B49 improved host immunity by increasing intestinal lysozyme activity, enhancing IL-8 expression, reducing TGF-β expression, and enhancing IgM levels in both serum and intestinal mucus. It also potentiated the phagocytic activity of peripheral blood lymphocytes. In addition, the B49 feeding group showed a significant increase in intestinal villus height. The challenge test with A. hydrophila demonstrated that the administration of P. acidilactici B49 effectively maintained intestinal barrier integrity, reduced gut inflammation, decreased pathogen load in the spleen, and improved survival rates in largemouth bass. In conclusion, the host-derived strain P. acidilactici B49 exhibited broad-spectrum antibacterial ability, biosafety, and intestinal colonization in largemouth bass. It effectively improved immune function, intestinal health, and resistance against A. hydrophila in the host. Therefore, P. acidilactici B49 is a promising probiotic for immunomodulation and disease control in largemouth bass aquaculture.