Background: As an early recognition of neonatal sepsis is important for triggering the initiation of treatment, this study was thus designed to assess the diagnostic performance and discrimination value of procalcitonin (PCT) in neonatal sepsis cases. Methods: This cross-sectional study, which was carried out at the Paediatric Intensive Care Unit of Hospital Universiti Sains Malaysia (HUSM) in Kelantan, Malaysia, had involved 60 neonates admitted for suspected sepsis. Sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV) and the area under receiver operating characteristics curve (AUC) for PCT were determined at initial presentation (0 h) as well as 12 h and 24 h after presentation in comparison to blood culture as the gold standard. Results: The study consisted of 27 (45.0%) male and 33 (55.0%) female neonates with a mean (SD) age of 76.8 (48.25) h. At cut-off PCT value of > 2 ng/mL, the sensitivity, specificity, PPV and NPV were 66.7%, 66.7%, 33.3% and 88.9% at 0 h. The respective parameters were 83.3%. 56.3%, 32.3% and 93.1% at 12 h and 83.3%, 52.1%, 30.3% and 92.6% at 24 h. AUC was 71.6%, 76.6% and 71.7% at 0 h, 12 h and 24 h. Conclusions: Diagnostic performance and discrimination values of PCT for diagnosis of neonatal sepsis varied with time of obtaining the blood samples. The PCT result at 12 h demonstrates the most optimal diagnostic performance and discrimination values.