Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen worldwide. However, data on MRSA prevalence in the African continent are scarce and nonexistent for São Tomé and Príncipe. In November 2010 and April 2012, a total of 332 individuals (258 patients and 74 health care workers [HCW]) from Hospital Dr. Ayres Menezes in São Tomé and Príncipe, were screened for S. aureus and MRSA carriage. Fifty-two persons (15.7%) were S. aureus nasal carriers out of which 14 (26.9%) were colonized with MRSA. MRSA isolates belonged to three clonal complexes: CC8 (PFGE type B-ST8-t064/t451-IVg/V), CC88 (PFGE E-ST88-t186/t786-IVa), and CC5 (PFGE K-ST5-t105-IVa/PFGE K-ST105-t002-II). A higher genetic diversity was found among methicillin-susceptible S. aureus (MSSA) isolates where 58.5% (n=24) belonged to four major lineages: PFGE type A-ST15-t084; PFGE C-ST508-t861 or related; PFGE D-ST152-t355 or related; and PFGE G-ST121-t159/t2304. Despite the common nonmultiresistant profile, 98% of the isolates harbored two or more virulence factors. Panton-Valentine leukocidine was detected in 36% of the isolates, all MSSA. S. aureus cross-transmission between HCW and patients in the pediatric and medicine wards and the detection of identical MRSA strains among patients in two different wards evidenced the need of implementation of additional infection control measures in this hospital.