Active surveillance culture (ASC) in neonatal intensive care units (NICUs) has been implemented to monitor horizontal transmission and choose appropriate antimicrobials when neonates experience late-onset infections (LOIs). To perform a cost analysis of ASC in the NICU, we evaluated data from a NICU ward in a Japanese community hospital. This was a retrospective study of neonates admitted to the NICU of Nara Prefecture General Medical Center, Nara City, Japan, from April 2012 to May 2017. We implemented biweekly ASC (nasal, stool and umbilical cultures) during this period. We assessed the detection rate for methicillin-sensitive and -resistant S. aureus (MSSA and MRSA) in each culture and reviewed the cases of LOI. Among the 785 cases, MSSA and MRSA were detected in 158 and 75 cases, respectively. Nasal culture was the most sensitive method for detecting MSSA and MRSA. Three cases benefitted from changing the empiric treatment based on previous ASC results, with an annual cost of ¥808,331 (£5773.80 or $7282.30) for ASC. The total cost per one benefited case during the study period was calculated as ¥1,392,126 (£9943.80 or $12,541.70). The total cost per benefited case in risk factor targeted ASC, nasal culture targeted ASC and MRSA-targeted ASC was ¥1,332,264 (£9516.20 or $12,002.30), ¥692,222 (£4944.40 or $6236.20) and ¥858,547 (£6132.50 or $7734.70), respectively. Considering the frequency of S. aureus detection and the cost of each culture, our ASC was modified to use only nasal cultures. This study suggests that the cost performance of ASC should be evaluated with consideration of the characteristics of each NICU.