Neonatal sepsis remains a major cause of morbidity and mortality. Therefore, early detection and initiation of appropriate empirical antibiotic therapy are crucial. The aim of this study was to describe the antibiogram of the neonatal intensive care unit at Grey's Hospital, a tertiary hospital in KwaZulu-Natal. This was a retrospective descriptive study, reviewing positive cultures from Grey's Hospital tertiary neonatal intensive care unit (NICU) in KwaZulu-Natal, South Africa for a 3-year period (01 January 2017 to 31 December 2019). All positive cultures from all sites were included. There were 1314 positive organisms cultured. Late-onset sepsis (89.3%) predominated over early-onset sepsis (10.7%). Blood was the source for 55.2% (725/1314) of positive cultures. Of the 1314 organisms cultured, 53.7% (706/1314) were Gram-positive, 45.7% (601/1314) were Gram-negative and 0.5% (7/1314) were Candida species. Klebsiella pneumoniae, 23.5% (313/1314) was the most frequent Gram-negative organism. It was noted to have high resistance to the unit's first-line antibiotic regimens; 99% were resistant to ampicillin and 92% resistant to gentamicin. Blood cultures yielded most positive results with a predominance of Gram-positive organisms and late-onset sepsis. A significant proportion of the cultured organisms were resistant to the first-line antimicrobials utilised in the unit, ampicillin and gentamicin. Ongoing surveillance on positive cultures is recommended to assess the effectiveness of the unit's current empirical antimicrobial guideline.