People working in health care services have an increased risk of being infected with methicillin-resistant Staphylococcus aureus (MRSA), though little is known about the prevalence in rehabilitation centers. This cross-sectional study investigated the MRSA prevalence in employees from different rehabilitation centers and aimed to identify risk factors for MRSA transmission. We invited all staff (i.e., with and without patient contact from 22 participating rehabilitation centers; n = 2499) to participate. Study participation included a questionnaire on personal characteristics, lifestyle, personal and occupational risk factors for MRSA and nasal swabs taken by the study team. In total, 1005 persons participated in the study (response: 40.2%). Only four participants carried MRSA (0.40 (95% CI 0.00–1.00) per 100). MRSA carriage did not seem to be occupationally related, as it was found in different occupations with and without direct contact with MRSA patients, as well as in different clinics with different indications and patient clientele. We could not find a clear association between MRSA carriage and potential risk factors due to the low number of cases found. Genotyping revealed the spa types t032 (Barnim epidemic strain) and t1223. Our results suggest a low point prevalence of nasal MRSA colonization in a non-outbreak setting in employees from rehabilitation centers.