<p>Aquaculture faces increasing challenges related to disease management and the need for sustainable alternatives to antibiotics that ensure productivity, fish welfare, and environmental sustainability. Probiotic bacteria, particularly lactic acid bacteria (LAB), have emerged as promising candidates for improving fish health through intestinal colonization, competitive exclusion of pathogens, and production of bioactive compounds. This study reports the phenotypic and genomic characterization of four <i>Pediococcus pentosaceus</i> strains (BE2, BE6, BE8, and BE9) isolated from the intestinal microbiota of freshwater fish (<i>Cichlasoma</i> spp.). Safety assessments revealed no hemolytic activity, coagulase production, or gelatinase activity, and antibiotic susceptibility profiles were consistent with international guidelines for probiotic candidates. The isolates demonstrated in vitro tolerance to acidic pH and bile salts, with viable counts decreasing by less than 2 log units under simulated gastrointestinal conditions. Adhesion assays using human Caco-2 cells showed approximately 30% adhesion efficiency. All strains exhibited growth on prebiotic substrates, including mannan oligosaccharides (MOS), fructo-oligosaccharides (FOS), and inulin (INU), with strain-specific preferences. Genomic analyses confirmed species-level identity and revealed biosynthetic gene clusters associated with the production of vitamins such as riboflavin, bacteriocins including penocin A and pediocin PA-1, alongside intrinsic resistance and stress response genes. Cell-free supernatants inhibited key aquaculture pathogens (<i>Streptococcus agalactiae</i>, <i>Aeromonas hydrophila</i>, and <i>Francisella orientalis</i>) in agar diffusion assays, suggesting antimicrobial potential mediated by bacteriocins identified in your genome. Therefore, the isolated strains exhibit promising functional and genomic characteristics, supporting their potential use as probiotics and components of synbiotic consortia for aquaculture applications.</p>