Aquaculture of popular freshwater species, Nile tilapia (Oreochromis niloticus), accounts for around 71% of the total global tilapia production. Frequent use of antibiotics for treating bacterial infections in tilapia leads to the emergence of antimicrobial resistance. To mitigate the issue, proper evaluation methods and control strategies have to be implemented. This study was aimed to analyze the antimicrobial resistance of bacterial isolates from the infected Nile tilapia cultured in freshwater. The recovered isolates were identified asPseudomonas entomophila, Edwardsiella tarda, Comamonassp, Delftia tsuruhatensis, Aeromonas dhakensis, A. sobria, A. hydrophila, A. lacus,Plesiomonas shigelloidesandVogesella perlucidathrough phenotypic and genotypic analyses. Using Primer-E software, Shannon Wiener diversity index of the isolates was determined asH' (loge) = 2.58. Antibiotic susceptibility test of the recovered strains through disk diffusion using 47 antibiotics, showed an elevated resistance pattern forAeromonas hydrophila,Pseudomonas entomophilaandComamonaswith higher multiple antibiotic resistance indexes (MAR index > 0.3). The minimum inhibitory concentration of antibiotics was > 256 mcg/ml for most of the resistant isolates. Meanwhile, all the recovered isolates were susceptible to amikacin, aztreonam, kanamycin, cefalexin, cefotaxime, levofloxacin, norfloxacin, piperacillin, and polymyxin-B.