Background: Neonatal sepsis is one of the leading causes of morbidity and mortality among preterm neonates, especially in developing countries such as India. Blood and CSF cultures are the gold standards for diagnosis and guidance of antimicrobial therapy in the identification of sepsis. However, the rise of multidrug-resistant (MDR) organisms presents a fresh challenge to the delivery of neonatal care. Objective: This study aimed to determine the microbial profile, trends in the resistance of organisms, and clinical outcomes of culture-proven late-onset sepsis among preterm neonates admitted to a tertiary NICU in North India. Methods: A retrospective cohort study of 2,187 preterm neonates admitted to NICU between January 2009 and December 2013. From that cohort, 693 had culture-proven sepsis (31.68%). Data were analyzed to obtain microbial profiles, antibiotic resistance, mortality rates, and NICU outcomes. Results: Gram-negative organisms predominated (71.74%); Klebsiella pneumonia (36.36%) and Escherichia coli (21.22%) were most frequently isolated. MDR was observed in 50.10% of the Gram-negative isolates. Carbapenem resistance was noted in 19.52%. Culture-positive sepsis correlated with longer NICU stay (mean: 19.74 ± 5.48 days) and mortality (28.13%). Conclusion: The present findings underscore the urgent necessity to strengthen antimicrobial stewardship and infection control measures against multidrug-resistant pathogens to enhance the neonate's outcome.