The study aims were to describe the frequency and dynamics of methicillin-resistant Staphylococcus aureus (MRSA) carriage among healthcare workers (HCWs), and to compare the molecular epidemiology of MRSA isolates from HCWs with those from patients with bacteremia. HCWs were interviewed and three nasal swabs were collected in a hospital in Lima, Peru, during 2009–2010. Consecutive S. aureus blood culture isolates from patients with bacteremia in the same hospital were also collected. SCCmec, multilocus sequence typing (MLST), and spa typing were performed. Persistent carriage was defined if having at least two consecutive cultures grown with S. aureus harboring an identical spa type. Among 172 HCWs included, the proportions of S. aureus and MRSA nasal carriage during first sampling were 22.7 % and 8.7 %, respectively. From 160 HCWs who were sampled three times, 12.5 % (20/160) were persistent S. aureus carriers and 26.9 % (43/160) were intermittent carriers. MRSA carriage among persistent and intermittent S. aureus carriers was 45.0 % (9/20) and 37.2 % (16/43), respectively. Fifty-six S. aureus blood culture isolates were analyzed, and 50 % (n = 28) were MRSA. Multidrug resistant ST5-spa t149-SCCmec I and ST72-spa t148-SCCmec non-typeable were the two most frequent genotypes detected among HCWs (91.7 %, i.e., 22/24 HCW in whom MRSA was isolated in at least one sample) and patients (24/28, 85.7 %). In conclusion, we found high proportions of MRSA among persistent and intermittent S. aureus nasal carriers among HCWs in a hospital in Lima. They belonged to similar genetic lineages as those recovered from patients with bacteremia.