Objective <p>Neonatal sepsis remains a major cause of morbidity and mortality, particularly among preterm and low-birth-weight infants requiring prolonged neonatal intensive care. This study aimed to describe the local epidemiology, microbiological profile, antimicrobial resistance patterns, and frequency of fulfillment of the European Medicines Agency (EMA) sepsis criteria among culture-proven neonatal sepsis episodes in a tertiary neonatal intensive care unit. The study was not designed to assess the diagnostic or predictive accuracy of the EMA criteria.</p> Methods <p>This retrospective single-center study included neonates with positive blood cultures admitted to the neonatal intensive care unit of Aydın Adnan Menderes University between January 2014 and December 2019. Infants with major congenital anomalies, contaminated cultures, or incomplete records were excluded. Demographic, clinical, microbiological, and laboratory data were analyzed, together with antimicrobial susceptibility results and EMA sepsis criteria.</p> Results <p>Among 5,515 hospitalized neonates, 190 infants with 212 culture-proven sepsis episodes were included. Of these episodes, 183 (86.3%) were late-onset neonatal sepsis, 8 (3.8%) were early-onset neonatal sepsis, and 21 (9.9%) were very late-onset neonatal sepsis. Among included infants, mean gestational age was 31.13 ± 4.54 weeks, and mean birth weight was 1613.61 ± 812.62&#xa0;g. Gram-positive organisms predominated (73.1%), with coagulase-negative staphylococci (CoNS) being the most frequent pathogens (64.6%); <i>Staphylococcus epidermidis</i> alone accounted for 35.8% of all episodes. Gram-negative organisms represented 23.1% of isolates, mainly <i>Klebsiella pneumoniae</i> (7.5%), and fungi accounted for 3.8%. Methicillin resistance was highly prevalent among CoNS, whereas all Gram-positive isolates remained susceptible to vancomycin. Among Gram-negative isolates, 7 of 16 <i>K. pneumoniae</i> strains were extended-spectrum beta-lactamase positive, and 2 isolates were meropenem resistant. EMA-positive sepsis criteria were fulfilled in 83.5% of episodes. Sepsis-related mortality was 7.9%. Episode-based mortality was higher in Gram-negative than Gram-positive infections.</p> Conclusion <p>Culture-proven neonatal sepsis in our NICU predominantly affected preterm infants and was mainly late-onset. CoNS were the leading pathogens, while Gram-negative organisms contributed substantially to severe and fatal infections. Continuous local surveillance and NICU-specific empirical antibiotic strategies are essential.</p>