Enterococcus faecium, a member of the Enterococcus genus, has emerged as a significant concern in aquaculture due to its potential pathogenicity and antibiotic resistance. This study aimed to investigate the molecular diagnosis and characterization of six different Enterococcus species isolated from the gastrointestinal tracts of the common carp (Cyprinus carpio) and tilapia (Oreochromis niloticus) and to evaluate their genetic diversity, antibiotic resistance profiles, and potential virulence factors. Among the isolates, 65.3% of the common carp and 60.8% of the tilapia were identified as E. faecium. All six species demonstrated the ability to metabolize various carbohydrates, indicating a broad metabolic capacity. Some species showed variability in their utilization of specific carbohydrates. For example, E. faecium and E. faecalis uniquely fermented adonitol, while E. avium and E. hirae were the only ones capable of fermenting D-arabitol. Additionally, Voges-Proskauer positivity was exclusively observed in E. faecalis. In terms of growth conditions, all species thrived at both 4°C and 45°C, except for E. faecalis, which failed to grow at 10°C. E. faecium and E. faecalis grew well at pH 9.6. Hemolysis tests revealed differences among the species: E. faecalis displayed beta-hemolysis, while E. gallinarum exhibited alpha-hemolysis. Motility was observed only in E. gallinarum, and esculin hydrolysis was exclusive to E. faecalis. Environmental adaptability varied among the species. E. avium showed limited growth in 6.5% NaCl, and some species exhibited little to no growth in 0.1% methylene blue milk. E. faecalis and E. faecium demonstrated survival at 60°C for 15 minutes, with E. faecium showing limited survival at 30 minutes, distinguishing them from the other species. The strains isolated from Cyprinus carpio and Oreochromis niloticus collected from local fish farms in Basrah City were confirmed as E. faecium through 16S rRNA gene sequencing. A PCR study using specific primers identified all isolates as E. faecalis.