Interference with antibiotic activity and its inactivation by bacterial modifying enzymes is a prevailing mode of bacterial resistance to antibiotics. Aminoglycoside antibiotics become inactivated by aminoglycoside-6'-N-acetyltransferase-Ib [AAC(6')-Ib] of gram-negative bacteria which transfers an acetyl group from acetyl-CoA to the antibiotic. The aim of the study was to disrupt the enzymatic activity of AAC(6')-Ib by adjuvants and restore aminoglycoside activity as a result. The binding affinities of several vitamins and chemical compounds with AAC(6')-Ib of Escherichia coli, Klebsiella pneumoniae, and Shigella sonnei were determined by molecular docking method to screen potential adjuvants. Adjuvants having higher binding affinity with target enzymes were further analyzed in-vitro to assess their impact on bacterial growth and bacterial modifying enzyme AAC(6')-Ib activity. Four compounds-zinc pyrithione (ZnPT), vitamin D, vitamin E and vitamin K-exhibited higher binding affinity to AAC(6')-Ib than the enzyme's natural substrate acetyl-CoA. Combination of each of these adjuvants with three aminoglycoside antibiotics-amikacin, gentamicin and kanamycin-were found to significantly increase the antibacterial activity against the selected bacterial species as well as hampering the activity of AAC(6')-Ib. The selection process of adjuvants and the use of those in combination with aminoglycoside antibiotics promises to be a novel area in overcoming bacterial resistance.