Background: Accurate and rapid diagnosis of neonatal septicemia is highly warranted because of high associated morbidity and mortality. Blood culture serves as a routine method for diagnosis of bacterial sepsis. However, it sometimes takes ≥ 72 hours for the results, with low sensitivity. Full automated blood culture method is superior to conventional methods in terms of speed and sensitivity. The polymerase chain reactionbased detection of 16S rRNA has reduced the laboratory turnaround time and has good sensitivity. Objectives: The aim of this study was to compare automated blood culture and amplification of 16srRNA gene by PCR in detection of aerobic bacterial infection in blood samples of hospitalized neonates with suspected neonatal sepsis and to determine the most common types of bacteria causing neonatal sepsis. Methodology: Blood samples collected from 40 neonates clinically suspected as neonatal sepsis were subjected to bacterial identification through automated blood culture by BacT/ALERT PF Plus Culture Bottles, and bacterial detection of 16S rRNA gene by PCR. Results: Out of 40 neonatal blood samples with suspected sepsis, 37(92.5%) neonates showed concordance between automated blood culture and PCR; 17(42.5%) showed positive results while 20 (50%) gave negative results by both methods. The remaining 3 cases (7.5%) were positive only by PCR. PCR sensitivity, specificity, positive and negative predictive values were 100%, 86.9%, 85% and 100% respectively. So, accuracy of PCR in relation to automated blood culture is 92.5%. The most common pathogens in cases of early onset neonatal sepsis were Klebsiella pneumoniae (7/15, 46.7%) followed by Streptococcus agalactiae (1/15, 6.6%) while the most common pathogens found in cases of late onset neonatal sepsis were Klebsiella pneumoniae and coagulase negative staphylococci (4/25,16% for each) then Staphylococcus aureus (1/25, 4%). Conclusion: 16S rRNA PCR showed accurate rapid diagnosis with higher sensitivity in diagnosis of neonatal septicemia. Klebsiella peumoniae was the main causative bacteria in early onset neonatal sepsis and late onset neonatal sepsis.