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Objectives The aim of our study was to determine the incidence of late onset sepsis and the most frequent microorganisms causing it in the Neonatal Unit (Neonatal Intensive Care Unit ie NICU and Level 2 nursery) at Hospital Cayetano Heredia, in Lima, Peru. Methods Descriptive and retrospective study. We reviewed all positive blood cultures and cultures of cerebrospinal fluid drawn from inborn patients beyond 72 hours of life, admitted to the NICU or our Level 2 nursery from January 2015 to December 2019. Results The incidence of late onset sepsis in our unit was 7.4% of admitted patients and 10.04 per 1000 live births. 234 episodes of LOS occurred in 204 patients through our study period. The incidence was higher in very low birth weight infants, reaching 36.2% and even higher in extremely-low birth weight infants (40.7%). The most frequent causative microorganisms were: coagulase-negative Staphylococcus, followed by Klebsiella spp. The most frequent cause of late onset sepsis in very low birth weight infants was gram negative bacteria (Klebsiella spp, being the most frequent microorganism). Conclusions Late onset neonatal sepsis is prevalent in our Neonatal Unit. It is important to know which are the most prevalent causative microorganisms in order to be able to choose an adequate antibiotic coverage and to design strategies to prevent them.