Sepsis in newborns is a leading cause of neonatal mortality in poor nations. The study aimed to identify the bacteria that cause sepsis in neonates in Egyptian hospitals, to characterize their resistance profiles, and to facilitate the selection of the most effective medication combination for multidrug-resistant bacteria. Between March 2019 to March 1, 2012, nine hundred eighty-nine blood samples were collected from newborns at Al Demerdash and Ain Shams University Specialized Hospital in accordance with established guidelines. The VITEK® 2 system was used to perform bacterial identification and antimicrobial susceptibility testing (AST). The results revealed that 51.4% of patients were positive for blood cultures, of which 60.25% were males and 39.8% were females. Late-onset sepsis was detected in 95% of positive cases, while early-onset sepsis was observed in only 5% of all positive cases. AST-ST01 card (bioMérieux) by VITEK 2 revealed that 96% of E. coli isolates were resistant to ampicillin, 91% were resistant to ampicillin / sulbactam, and 83% were resistant to ceftazidime and cefotaxime. The blaCTXM15 gene expression was analyzed quantitatively using real-time RT-PCR and shown to be significantly higher in MDR E. coli isolates (100 %) than in susceptible isolates, with an average change of 8.0458-fold versus 1.7581. In conclusion, E. coli was the most common cause of neonatal sepsis in Egyptian neonatal intensive care units (NICUs). Applying suitable antibiotic management systems in conjunction with infection prevention and control measures might aid in this attempt.