Timor-Leste has a significant burden of infectious diseases, but historically has had limited diagnostic capacity and availability of microbiology data on human health. Recent developments in the diagnosis and reporting of key pathogens including Staphylococcus aureus have allowed better understanding of the burden of key infectious diseases and their impact on the population of Timor-Leste. A prospective observational study on clinical isolates of S. aureus received at Direcção Geral Laboratório da Saúde (DGLS) in Dili, Timor-Leste between January 2020 and July 2020 was performed. Clinical samples were obtained from patients living in 11 out of the 13 municipalities in Timor-Leste. Standard microbiology culture, identification, and antimicrobial susceptibility testing (AST) were performed, and clinical and demographic data was collected on laboratory-confirmed S. aureus isolates. A total of 59 clinical isolates of S. aureus were identified. Most patients in our study were found to have community-acquired S. aureus (75%), whilst the remaining 25% were hospital-associated infection (HAI). A quarter of all S. aureus isolates were resistant to methicillin. This is the first comprehensive description of S. aureus infections in Timor-Leste. The high MRSA rates identified in this study can be used to better inform guidelines for empirical treatment of S. aureus infection. Continuous investment in detecting clinically important pathogens and understanding their susceptibility profiles is critical for the development of treatment guidelines and antibiotic stewardship activities.