Introduction: Antibiotic resistance is an evolving threat of our time, influencing public health and provision of health care worldwide. Antibiotic resistance not only can affect the individuals at any stage of life but also the veterinary and agriculture industries. Developing new antibiotics or β-Lactamase Enzymes Specific Inhibitors (BESI) are two main strategies to overcome the problem of multi-drug resistant bacteria. Aim: Our main goal was to evaluate the possibility of our developed BESI (Inhibitor-X) to inhibit β-Lactamase enzymes of some of the most multidrug resistant Gram Positive and Gram Negative bacteria. Materials and methods: During the past 18 years, we tried to develop and evaluate numerous products functioning as BESIs.Reference strains of Gram Positive bacteria (Staphylococcus aureus, MRSA, ATCC® 43300) (WHO high priority) and Gram Negative bacteria (Klebsiella pneumonia ATCC ® BAA-1144 (AMPC weak positive), Klebsiella pneumoniae ATCC® 700603, Klebsiella pneumoniae ATCC® 700603 (ESBL), Enterobacter cloacae ATCC® BAA-1143 (AMPC highly positive) and Pseudomonas aeruginosa (ATCC® 27853) (WHO critical priority) were obtained commercially. The bacteria were cultured in Mueller-Hinton agar at 35°C and antibiogram tests were performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Antimicrobial activities were investigated by minimum inhibitory concentration (MIC) and Kirby-Bauer disc diffusion test. MICs were determined in triplicates by the broth microdilution MIC method recommended by CLSI.The antibiotic concentrations ranged from 0.03125-16 µg/ml. The titer plates were inoculated with bacteria having 0.5 McFarland turbidity, and incubated aerobically at 35°C for 24 hours. The synergistic effect was measured using the following: , where ɑ is the inhibitory concentration of the antibiotic and ɓ is the inhibitory concentration of the antibiotic in the presence of Inhibitor-X. Cytotoxicity of Inhibitor-X was evaluated by mouse embryo assay and in cell culture.Results: Inhibitor-X in combination with cefotaxime resulted in a significantly large inhibition zone (0 vs. 25±1 mm, mean±SEM, n= 138, P<0.001) (Fig.1B&K, Fig.2). Similar results were obtained using Inhibitor-X + ceftriaxone (10±1 vs. 15±2 mm, mean±SEM, n= 97, P<0.01) (Fig.2). Inhibitor-X showed also excellent synergistic action in combination with cefepime to inhibit MRSA, ATCC® 43300 (0 vs. 24±1mm, mean±SEM, n=111, P<0.001) (Fig. 2D&E). The effect of Inhibitor-X with cefotaxime against Enterobacter cloacae ATCC® BAA-1143 was highly improved (0 vs. 15±1 mm, mean±SEM, n=64,P<0.001)(Fig. 4G vs. 4H). The effect of Inhibitor-X with cefotaxime was also improved against Klebsiella pneumonia ATCC ® BAA1144 (25±1 vs. 33±1 mm, mean±SEM, n=76)(Fig.3)(Fig.4A vs. 4B), Klebsiella pneumoniae ATCC® 700603(24±1 vs. 30±1 mm, mean±SEM, n=91) (Fig.3) and Pseudomonas aeruginosa ATCC® 27853 (18±1 vs.34±1 mm, mean±SEM, n=63)(Fig.3)