Extensively drug-resistant (XDR), multidrug-resistant (MDR) and pandrug-resistant (PDR) Gram-negative microorganisms (GNBs) are considered a significant global threat. β-lactam and aminoglycoside combinations and imipenem:cyclodextrin inclusion complexes were studied for the treatment of lethal GNBs. This is because of the broad empiric coverage of the two drugs and their possession of different spectra of activity. Two cyclodextrins (β- and hydroxy propyl β-cyclodextrins) were utilized for inclusion complex formation with imipenem using the physical and kneading methods. In silico investigation using the molecular docking and Fourier-infrared spectroscopy (FTIR) were employed to estimate binding constant and confirm complex formation, respectively. The in vitro effects of amikacin and imipenem combination in comparison to the effect of imipenem-β- and hydroxy propyl β-cyclodextrin (CD) complexes against Klebsiella spp. and Acinetobacter baumannii were studied. The isolated microorganisms’ antimicrobial responsiveness to various antibiotics (19 antibiotics) was evaluated. It was found that piperacillin/tazobactam and gentamycin (resistance rates were 33.3% and 34%, respectively) were the most effective antimicrobials. The in vitro studies have been performed by the checkerboard technique and time-killing assay. The studied combination of amikacin and imipenem showed a substantial drop in bacterial count (p < 0.05). The in vitro studies demonstrated a synergism for the investigated combination. Conventional PCR was used in molecular studies to identify the resistance genes bla IMP and aac (6′)-Ib. The blaIMP and aac (6′)-Ib were recorded in 38.2% and 3.6% of the studied isolates, respectively. The in vitro studies showed synergistic effects among the tested antibiotics with FICIs of ≤0.5. Finally, the study compared the reduction in bacterial count between the tested antibiotic combinations and imipenem:CD physical and kneaded mixtures. Imipenem:CD inclusion complexes demonstrated a significant bacterial count reduction over the antibiotic combination. These results highlight the emerging role of CDs as safe biofunctional excipients in the combat against superbug bacterial resistance.