β-lactam antibiotics are the most extensively used antibiotics to treat a variety of infections due to their safety, high selectivity, and efficacy. However, the main cause of antibiotic resistance is due to the misuse of antibiotics uptake which stimulated bacteria to produce β-lactamases. Therefore, there is an essential need for the development of novel β-lactam/β-lactamase inhibitor combinations. In this respect, 186 Actinomycetes isolates were screened for β-lactamase inhibitors (BLIs) using disc diffusion and modified Iodometric bioassays. Two Egyptian Streptomyces isolates (S.t-158 & S.t-6) were selected for their ability to exhibit BLIs. The Egyptian isolates S.t-158 and S.t-6 were molecularly identified by 16s rRNA gene sequencing with 100% and 98.9% nucleotide similarity to S. rochei and S. clavuligerus, respectively. β-Lactamase inhibitory protein (BLIP) gene (600 Bp) was detected in the Egyptian S. clavuligerus strain. In-silico docking of β-lactamase enzyme (KPC-2) against four sets of selected ligands was performed using Molecular Operating Environment to investigate possible binding interactions of selected bioactive compounds within the enzyme active site. Harmine, Borrelidin and SRB-2 showed high binding energy values of -5.699, -6.531 and -5.631, respectively. These compounds form hydrogen bonds with KPC-2 active site residue-Ser70 or -Ser130 and prevent it from attacking β-lactam antibiotics, thus inactivating it. The BLIs could be useful for synergic therapy with β-lactams antibiotics to overcome the β-lactamase resistance.