In the blood culture procedure for neonatal sepsis, time to positivity (TTP) reflects the pathogenic bacterial load and the time required for empirical antibiotic regimen administration prior to definitive treatment. This study aims to identify the differences in TTP among causative pathogens and its predictive value for the overall survival of neonates with sepsis at a tertiary healthcare center in Indonesia. Methods: A retrospective cohort study was conducted from January 2020 to August 2022 at Dr. Soetomo General Hospital, Surabaya, Indonesia. Neonates with blood culture-proven neonatal sepsis were included in the analysis. TTP was defined as the time between the acceptance of a blood culture specimen from the neonatal intensive care unit and reports of positive culture growth by the laboratory. Results: Across 125 cases, the median TTP was 58.1 hours (IQR = 24.48). Blood cultures were positive within 48 hours for 41.6% of cases, 72 hours for 86.4%, and 96 hours for 98.4%. A significantly shorter TTP was exhibited by the three major gram-negative organisms (Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter cloacae) compared to coagulasenegative Staphylococci. The neonatal sepsis mortality rate was 49.6% during the study period. In the Cox multivariate regression model, a shorter TTP was an independently predicted mortality in the entire cohort (hazard ratio (HR) = 0.985, 95% CI: 0.973– 0.998) and the gram-negative sepsis cohort group (HR = 0.983, 95% CI: 0.968–0.999). Conclusions: TTP predicts different causative pathogens and the overall survival of neonatal sepsis cases at a tertiary healthcare facility in Indonesia