Objective: To retrospectively determine if a negative 16S ribosomal RNA (rRNA) polymerase chain reaction (PCR) (PCR(-)) could lead to a decrease in the number of antibiotic doses and neonatal intensive care unit (NICU) length of stay (LOS) for infants admitted to the NICU for presumed early-onset sepsis (EOS) with negative blood culture results (BC(-)). Study design: Analysis included 419 infants, greater than 35 weeks gestational age, with PCR(-), BC(-) and LOS>48h. Both the investigators and clinical care team were unaware of the PCR results. The actual number of antibiotic doses (AAD) administered was compared to an estimated number of antibiotics doses (EAD) that would have been given until PCR(-) results were available by 18h. The number of antibiotic doses saved was calculated as (AAD-EAD). The actual NICU LOS in hours (aLOS) for a subset of infants who remained in the hospital primarily for antibiotic therapy was compared to an estimated LOS (eLOS) if infants with PCR(-) were discharged from the NICU when clinically stable. The number of hours saved was calculated as (aLOS-eLOS). Results: Approximately eight antibiotic doses and 85 NICU hours per infant could be saved using PCR(-) results available at 18h. Conclusions: Use of 16S rRNA PCR could decrease the number of antibiotics doses and NICU LOS for infants admitted for EOS. This may facilitate: (1) earlier NICU discharge; (2) parental satisfaction; and (3) decreased health care costs.