BACKGROUND Antibiotic therapy for neonatal Staphylococcus aureus bacteremia (NSAB) is recommended for at least 14 days. However, this recommendation is based on very limited data. This study aimed to compare NSAB relapses between patients treated for less than 14 days versus 14 days or more in neonatal intensive care units. METHODS This retrospective study was conducted in 3 neonatal intensive care units. All patients with a positive blood culture for S. aureus from January 2010 to December 2022 were included. Relapse was defined as the identification of S. aureus in a blood culture within 3 months after cessation of antibiotic therapy. RESULTS Of 169 patients with positive blood culture for S. aureus, 134 patients were included in the analysis. Fifteen (9%) were excluded because cultures were considered contamination, 19 (11%) because of death before completing treatment, and one because of missing data. The median gestational age was 28 weeks. One hundred patients (75%) had a central venous line. The median duration of antibiotic therapy was 8 days (interquartile range: 6-9) in the <14-day group, and 19.5 days (interquartile range: 17-23) in the ≥14-day group. A relapse occurred in 15 patients: 11 (12%) in the <14-day group (n = 94) and 4 (10%) in the ≥14-day group (n = 40), with no significant difference between the groups (P = 0.775). CONCLUSIONS In this cohort, no increased risk of NSAB relapse was observed with treatment durations of less than 14 days. Further trials are necessary to determine the optimal treatment duration for NSAB.