Background: The carrier state of methicillin-resistant Staphylococcus aureus is pointed as an infection predictor and a factor for environmental and person-to-person dissemination, including health service workers. These, when colonized are commonly associated to outbreaks. Objective: Analyze the prevalence of Staphylococcus aureus in saliva of workers at a university hospital. Methodology: Epidemiologic longitudinal study carried out in Curitiba, Paraná, Brazil, with 486 workers between April 2006 and June 2008. Three saliva samples were collected and a data collection instrument was applied. Staphylococcus aureus were isolated from the clinical specimen and characterized by phenotypes. The data from the instrument and the laboratory results were organized and processed with EPI-Info software and analyzed via descriptive statistics. Results: Among the healthcare workers studied, 60.9% were colonized by Staphylococcus aureus in saliva; of those, 67.9% were transitory carriers and 32.1% were persistent carriers; the prevalence of meticillin-resistant Staphylococcus aureus (MRSA) among the isolated cases was 15.7%. The average prevalence of MRSA was 12.7% and higher among nurses’ aides (21.4%) and cleaning aides (20.6%) and lower among nurses (4.5%) and doctors (5.9%). Conclusions: Healthcare workers presented high prevalence of Staphylococcus aureus in saliva, indicating the mouth as an important body site to investigate colonization by MRSA and a potential source to its dissemination.