There is a paucity of data on Staphylococcus aureus epidemiology in Africa. Prevalence of nasal carriage and genetic diversity of S. aureus were determined among hospital staff (HS) and inpatients (IP) at the largest hospital in Ghana. In total, 632 nasal swabs were obtained from 452 IP and 180 HS in the Child Health Department (CHD) and Surgical Department (SD). S. aureus carriage prevalences were 13.9% in IP and 23.3% in HS. The chance of being a carrier was higher in HS (P=0.005) and IP staying ≤7 days in hospital (P=0.007). Resistance to penicillin (93%), tetracycline (28%) and fusidic acid (12%) was more common than for other agents (<5%). A higher chance of multidrug-resistant S. aureus carriage was observed among IP compared with HS (P=0.01). High genetic diversity was shown by spa typing, with 55 spa types found among 105 isolates; the predominant spa types were t355 (10%) and t084 (10%). MRSA was detected in six IP with an overall carriage prevalence of ca. 1.3%, but not in HS. All three MRSA isolates from SD belonged to ST88-SCCmec IV, and two of them displayed the same spa type and antibiograms; three MRSA isolates from CHD belonged to distinct lineages (ST88-SCCmec IV, ST8-SCCmec V and ST72-SCCmec V). Altogether, these data indicate a high diversity of S. aureus, low levels of MRSA carriage, and a higher chance of nasal carriage of multidrug-resistant S. aureus among IP compared with HS in this hospital.