Purpose: To identify the frequency of bacterial isolates in early-onset neonatal sepsis (EONS) and their antimicrobial resistance pattern. Methods: A retrospective study of EONS was conducted at the Beni Suef University Hospital from September 2008 to September 2012. A case of EONS was defined as an infant who had clinical signs of infection or who was born to a mother with risk factors for infection, and in whom blood culture obtained within 72 hours of life grew a bacterial pathogen. Results: Of 673 neonates screened, there were 138 positive blood cultures (20.5%) (confirmed EONS). Of the recovered isolates, 86.2% were gram-negative pathogens. Klebsiella pneumoniae (42.8%), Enterobacter cloacae (22.5%), and Escherichia coli (13.8%) were the commonest isolated organisms. The most common gram-positive microorganism was Staphylococcus aureus accounting for only 12 isolates (8.7%). All Klebsiella isolates and 93% of Enterobacter isolates were resistant to ampicillin. Gram-negative pathogens had the maximum overall sensitivity to imipenem, cefepime, and ciprofloxacin; whereas, gram-positive isolates were most susceptible to vancomycin, imipenem, and piperacillin. Conclusion: K. pneumoniae was the predominant causative bacteria of EONS followed by E. cloacae and E. coli. There was a high resistance to ampicillin. Imipenem had the maximum overall activity against the causative bacteria. Continuous surveillance is needed to monitor the changing epidemiology of pathogens and antibiotic sensitivity.