From the perspective of gold nanoparticles (GNPs)' potential antibacterial applications, we demonstrate the fabrication, characterization, and effective antimicrobial activity of gentamicin (GM) and kanamycin (KAN) dynamically loaded to glucose-capped gold nanoparticles (g-GNPs). Infra-red (FT-IR) spectroscopy analyzed the synthesized GNPs and g-GNPs with encapsulated antibiotic drugs. Various spectroscopical methods characterized g-GNPs and drug-loaded nanoparticles. A minimum inhibitory concentration (MIC) and active curves in the Klebsiella pneumonia strain were used to evaluate the antibacterial activity of aminoglycoside-loaded g-GNPs, and the results showed that the nanoparticles had an appropriate antimicrobial effect on the clinical strain of the bacteria. There were bacteriostatic effects and an inhibiting effect on the growth of bacteria at doses of 33 to 65 µg/ml for each GM@g-GNPs and KAN@g-GNPs. With zones of Inhibition (ZOI) of 27 and 29 mm, g-GNPs-loaded antimicrobial medications demonstrated more antibacterial activity in well diffusion experiments than free nanomaterials and antimicrobial drugs, with ZOI of 21.00 and 10.00 mm, respectively. GM and KAN-loaded g-GNPs were examined using crystal violet assay for their antibiofilm activity. Findings suggested that the concentration of nanoparticles and anti-biofilm activity were directly linked. The cell membrane integrity was assessed in g-GNPs loaded with GM and KAN, and the results showed that nucleic acids and proteins released into the environment were significant. The inhibitory effects of GM@g-GNPs and KAN@g-GNPs on bacterial efflux pump (EP) was assessed, and the result displayed that all strains were sensitive to moderate concentration of NPs and highly sensitive to concentrations of 0.6 and 0.9 µg/ml of ethidium bromide (EtBr) and 64 to 128 µg/ml of GM@g-GNPs and KAN@g-GNPs. The findings demonstrate that all strains were responsive to moderate nanoparticle concentrations.