Background: Coagulase-negative Staphylococcus (CoNS), is the most frequent pathogen causing late onset sepsis (LOS) in neonatal intensive-care units (NICUs). Technical difficulties hinder blood culture (BC) collection and obtaining only one culture before initiating antibiotic therapy is common practice. We sought to assess specific clinical information and CoNS cultures for the diagnosis of true bacteremia in the NICU. Methods: This historical cohort study was conducted in NICUs at Hadassah-Hebrew University Medical Center, Jerusalem, Israel. Clinical and laboratory data in every CoNS bacteremia were collected and compared between bacteremia groups: true-positive – two positive BCs; contaminant – one positive BC out of two; undefined – one BC obtained and found positive. Results: During 3.5 years, CoNS was isolated in 139 episodes. True-positive was identified in 44/139 (31.7%), contaminant in 42/139 (30.2%) and in 53/139 (38.1%) the event was undefined. Vancomycin treatment was more frequent in the true-positive and undefined groups than the contaminant group (100%, 90.6% and 73.8% respectively, p=0.001); treatment was also prolonged in these two groups (p<0.001). No clinical variables were associated with true bacteremia on multivariable analysis. Conclusions: Diagnosis should definitely be based on at least two positive BCs, despite objective difficulties in obtaining BCs in neonates.