Abstract Background Enterococci are intrinsically resistant to clinically achievable concentrations of aminoglycosides. However, high-level resistance to aminoglycosides (HLAR) is primarily due to the acquisition of genes encoding aminoglycoside-modifying enzymes (AMEs). Aminoglycosides along with cell wall inhibitors are given clinically for treating enterococcal infections. The current study was conducted to investigate the rate of HLAR and to determine aminoglycoside resistance encoding genes profile in enterococcal isolates from different clinical specimens. Results From 120 Enterococcus species, 50 (41.7%) enterococcal isolates were proven to have HLAR, 78% (39/50) have high-level gentamicin resistance (HLGR), and 74% (37/50) were high-level streptomycin-resistant (HLSR). HLGR isolates carried aminoglycoside modifying gene aac (6′)-Ie-aph (2′)-Ia in 26/39 (66.7%) of isolates, whereas 32/37 (86.5%) of HLSR carried aph (3′)-IIIa gene and were observed in E. faecalis, E. faecium, E. gallinarum, and E. casseliflavus. The aph (2′)-Ib, aph (2′)-Ic, and aph (2′)-Id that encode HLGR could not be detected. Conclusions The high detection rate of HLAR among the studied Enterococcus species and the coexistence of HLGR and HLSR strains provide crucial insights to the necessity of routine testing for HLAR in the microbiology lab. The main AME genes among HLGR and HLSR enterococci were aac (6′)-Ie-aph (2″)-Ia and aph (3′)-IIIa, respectively.