Background <p><i>Candida albicans</i> is the primary pathogen responsible for candidemia, particularly in hospital settings, and is often associated with outbreaks in neonatal intensive care units (NICUs). In this study, we aimed to investigate the transmission dynamics and identify the potential source of a <i>C. albicans</i> candidemia outbreak in an NICU, focusing on neonates at high risk of infection.</p> Methods <p>This retrospective, single-center study focused on analyzing 10 neonates with candidemia and 23 <i>C. albicans</i> strains, including 6 isolates obtained from the environment and staff members in a neonatal ward. Whole-genome sequencing (WGS) was employed to trace the outbreak of candidemia in neonatal wards and reconstruct the transmission path of infection based on epidemiological data.</p> Results <p>70% (7/10) of neonates had either extremely or very low body weight as well as catheter-related bloodstream infection. Multilocus sequence typing revealed that the isolates could be categorized into three sequence types (STs), including two novel STs, ST3601 and ST3602, with ST3601 being predominant (82.6%, 19/23). Genome SNP/InDel variation and phylogenetic analyses revealed that the outbreak isolates (C1-C17, C19, and C22) formed a distinct cluster, exhibiting a reduced mutation distance, as evidenced by both the phylogenetic tree and heatmap analyses, and shared a close genetic relationship with each other, suggesting a common source of infection.</p> Conclusions <p>Among neonates with extremely or very low body weight, central venous catheter-related candidemia was the most frequent, and the contaminated hands of medical staff were likely a medium of transmission or the staff was contaminated with the <i>candida</i> from the neonates. This study highlights the utility of WGS in detecting and understanding hospital outbreaks, helping to trace transmission routes and improve infection control.</p> Clinical trial number <p>Not applicable.</p> Graphical Abstract <p></p>