Abstract Background We estimated the nationwide burden of nosocomial S. aureus bloodstream infection (SA-BSI), a major cause of nosocomial infection, in South Korea. Methods To evaluate the nationwide incidence of nosocomial SA-BSI, cases of SA-BSI were prospectively collected from 22 hospitals with over 500 beds over 4 months. Data on patient-days were obtained from a national health insurance database containing the claims data for all healthcare facilities in South Korea. The additional cost of SA-BSI was estimated through a matched case–control study. The economic burden was calculated from the sum of the medical costs, the costs of caregiving and loss of productivity. Results Three hundred and thirty nine cases of nosocomial SA-BSI were included in the study: 254 cases of methicillin-resistant SA-BSI (MRSA-BSI) and 85 cases of methicillin-susceptible SA-BSI (MSSA-BSI). Death related to BSI occurred in 81 cases (31.9%) of MRSA-BSI and 12 cases (14.1%) of MSSA-BSI. The estimated incidence of nosocomial MRSA-BSI was 0.12/1,000 patient-days and that of nosocomial MSSA-BSI, 0.04/1,000 patient-days. The estimated annual cases of nosocomial BSI were 2,946 for MRSA and 986 for MSSA in South Korea. The additional economic burden per case of nosocomial SA-BSI was US $20,494 for MRSA-BSI and $6,914 for MSSA-BSI. Total additional annual cost of nosocomial SA-BSI was $67,192,559. Conclusion In view of the burden of nosocomial SA-BSI, a national strategy for reducing nosocomial SA-BSI is urgently needed in South Korea.