Introduction In developing countries, sepsis and associated mortality rates in neonatal patients is a serious concern. To improve the outcomes and mortality posed by sepsis, physicians need to know the local epidemiology of the microbial pathogens and their resistance patterns to antimicrobial agents. Therefore, our aim was to determine the frequency of early-onset neonatal sepsis (EONS) following prolonged rupture of membranes (PROM). Materials and methods After approval from the ethical review committee, this cross-sectional study was conducted at a tertiary care hospital of a developing country, and informed consent was taken from patients’ parents. All neonates born to a mother with PROM after 24 weeks of gestation up to seven days of life were included. Demographic features, signs of sepsis, blood culture results, and laboratory markers of sepsis were recorded. All data were analyzed by using IBM SPSS Statistics for Windows, Version 20.0 (IBM Corp., Armonk, NY). Results A total of 124 patients were enrolled in the study. Seven neonates (5.6%) developed EONS and positive cultures were seen in four neonates (3.2%) with a maternal history of PROM. The organisms identified in cultures were Klebsiella pneumonia, group B streptococcus, Staphylococcus aureus, and Streptococcus species in EONS caused by prolonged PROM. Conclusions Early recognition of risk factors, recognition of clinical conditions with prompt laboratory screening for infection, and early establishment of empirical antibiotic treatment are effective preventive measures. Such approaches would be a secure and efficient strategy, particularly in developing countries.Copyright © 2020, Rathore et al.