Gram-negative bacteria have been established in Egypt to have high rates of antimicrobial resistance. A major threat to human health exists in every country due to the emergence and spread of carbapenem-resistant gram-negative bacteria. A total of 127 Gram-negative bacterial isolates were identified and their antibiotic susceptibility testing were performed. They include 100 K. pneumoniae and 27 E. coli isolates. Phenotypic identification of carbapenemase production was confirmed by Modified Hodge test (MHT) and Modified Carbapenem Inactivation Method (mCIM). The blaOXA-48 gene was detected using two variant primers (OXA-438 and OXA-743) conventional polymerase chain reaction. The antimicrobial susceptibility test for K. pneumoniae and E. coli showed 100% resistance to ampicillin, amoxicillin, pipracillin/tazobactam, cefotaxime, cefepime, ceftazidime, imipenem, meropenem, ciprofloxacin, ofloxacin and nitrofurantoin. K. pneumoniae also possessed resistances reached 91% to gentamycin, 86% to co-trimoxazole and 79% to amikacin. On the other, 100% of E. coli isolates were resistant to co-trimoxazole. Additionally, they exhibited resistance to gentamycin (81.5%) and amikacin (77.8%). Genotypic study using multiplex PCR for detection of the blaOXA-48 gene (by two variant primers: OXA-438 and OXA-743) revealed that among the 100 K. pneumoniae isolates, 22% and 18% were detected using OXA-438 and OXA-743, respectively. Similarly, the 27 carbapenem resistant E. coli isolates revealed the presence of 7.4% of both OXA-438 and OXA-743 primers.