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Background: Enterobacteriaceae, common pathogens responsible for urinary tract infections are known to be susceptible to aminoglycosides. The emergence of resistant pathogens complicates antimicrobial regimen and becomes a challenge for clinicians. The aim of this study was to evaluate the susceptibility of clinically isolated Enterobacteriaceae to isepamicin and amikacin using disc diffusion method. Materials and Methods: A total of 234 Enterobacteriaceae isolates was collected and examined. Antimicrobial susceptibilities to gentamicin, amikacin, and isepamicin were assessed using disc diffusion method. The production of extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase was also tested. The susceptibilities of the pathogens to isepamicin and amikacin were evaluated. Results: Two hundred and thirty-four Enterobacteriaceae isolates were found to be more susceptible to amikacin and isepamicin than to gentamicin. Of the isolates, 39 (16.7%) produced ESBL and 41 (17.5%) harbored AmpC β-lactamase. The results revealed that amikacin and isepamicin exerted excellent antibacterial activity (94% vs. 93.6%) against all tested isolates. Isepamicin was effective against 89.7% ESBL-producing isolates and 92.7% of AmpC-producing isolates. The susceptibility to amikacin and isepamicin established by the disc diffusion method was mostly consistent with the overall agreement estimated 99.6%. Conclusions: Isepamicin showed excellent activities against infections caused by Enterobacteriaceae, including strains harboring ESBL or AmpC beta-lactamase. The susceptibility of tested isolated to isepamicin measured by disc diffusion method is comparable to that of amikacin.