Background: Sepsis in the neonatal intensive care unit (NICU) remains one of the most significant causes of morbidity and mortality, especially for preterm. Multi-drugresistant-organisms (MDROs) are emerging as important pathogens that cause neonatal sepsis in NICU. Objective: to review the epidemiology of the microorganisms implicated in neonatal sepsis while shedding the light on the percentage of Multipledrug-resistant (MDR) and Extensive-Drug-Resistance (XDR) microorganisms in addition to investigating their antibiotic susceptibility pattern. Methodology: This is a cross-sectional prospective study of a 24-month duration including data from cultureproven neonatal sepsis patients admitted at NICU, from Fayoum University Pediatric Hospital. Results: Klebsiella species was the most isolated organism from blood (46%), and 87.6% of isolates were MDR organisms (332/379). The resistance pattern was as follows: 66.5% of resistance owed to Gram-negative bacilli; of them 52% were XDR, 13.2% were MDR, 0.8% were Pan drug Resistant and 0.5% were Difficult-to-Treat (DTR)-Pseudomonas species. Gram-positive cocci were responsible for 21.1 % of MDR; 20.8% MRSA and 0.3% VRE. Total MDR accounted for 34.3% of isolates. The isolates showed significant resistance to most tested antibiotics (p<0.05) (doxycycline, tetracycline, amikacin, cefoxitin, meropenem, imipenem, ertapenem, piperacillintazobactam, and ampicillin-sulbactam. Significant sensitivity was detected to linezolid, vancomycin, and tigecycline. Conclusion: There is an alarming increase in the resistance rates among cases of neonatal sepsis. The application of an antibiotic stewardship program is essentially needed to stop the dramatic increase in antibiotic resistance and fight the development of MDROs.