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To determine the bacteriological pattern and antibiotic susceptibility of bacterial isolates causing neonatal sepsis in Qena University Hospitals and compare polymerase chain reaction (PCR) and blood culture results in a trial for rapid diagnosis. Blood samples from 75 clinically suspected cases of neonatal sepsis were subjected to identification of bacteria and determination of their antibiotic sensitivity through blood culture, and rapid detection of 16S rRNA and the uidA gene (to confirm the presence of ) by PCR from extracted bacterial DNA. Most patients were preterm (64%) and low birth weight (LBW) (68%). In total, 42.7% presented with early onset sepsis (EOS). LBW was significantly associated with EOS ( -value=0.03). Although the blood culture and PCR results were similar in EOS, the PCR results were significantly higher than those of blood culture in detecting bacteria (85.3% vs 68%, respectively, -value=0.001). Blood culture showed 100% specificity. The most common pathogen was (86.2%) in EOS and . (45.5%) in late-onset sepsis (LOS) ( -value=0.001 and 0.02, respectively). The most effective antibiotics against Gram-negative bacteria were ofloxacin, ciprofloxacin, imipenem, and amikacin, while vancomycin, oxacillin, and imipenem were the most effective antibiotics against Gram-positive bacteria. EOS was mainly caused by , while LOS was mainly caused by . The 16S rRNA PCR showed higher sensitivity with rapid and accurate diagnosis. Blood culture is the most suitable method for antimicrobial sensitivity testing.