Background: Hospital acquired infections are still a major cause of morbidity and mortality among neonates and children admitted to intensive care units Objectives: To determine the prevalence and clinical distribution of Multidrug resistant organisms (MDROs) in neonatal intensive care unit (NICU) and pediatric intensive care unit (PICU) of Beni-Suef University Hospital. Methodology: A cross-sectional study included 160 patients diagnosed with sepsis, 80 patients admitted at NICU and another 80 patients at PICU. Blood, sputum, urine were collected from each patient, to perform; CBC, CRP, culture and sensitivity. Identification of isolates was conducted by the various conventional methods. Thereafter, antimicrobial sensitivity tests were conducted for each isolate to detect MDROs. Results: Most of the isolates were MDROs (85.8%%) with high statistical significant difference regarding their frequencies. Most common Gram-negative isolate was klebsiella pneumoniae representing 36.7% of isolates. Candida albicans were significantly more frequent among PICU cases (p-value <0.05). MDR risk factors revealed that: length of stay (LOS) at the present ICU or LOS in another ICU before the current one were associated risk factors (p-value = 0.00001, 0.05 respectively). MDR Gram-negative isolates showed high resistance to ampicillin-sulbactam (98.8%) and were most sensitive to polymyxin (79.1%). In MDR Gram-positive yields, vancomycin (93%) and linozolide (100%) were the most effective, whilst, resistance was evident against ampicillin (93%). All Candida spp. isolates were most sensitive to amphotericin (93.1%) and most resistant to fluconazole (62%). The overall mortality rate was 32.5%: NICU deaths represented 52.5% and PICU deaths 12.5% with a significant difference between NICU and PICU. Conclusion: Prevalence rate of MDROs is extremely high among NICU and PICU patients even against newer categories of antibiotics, so, more strict infection control program should be applied.