In several European countries, diagnosis of nephropathia epidemica, a mild form of haemorrhagic fever with renal syndrome (HFRS) caused by Puumala-virus infection, has increased over the past 10–20 years. In Switzerland, despite its geographical proximity to regions with epidemic outbreaks in Germany and France, infections are detected only sporadically. To estimate the actual prevalence and potential risk factors of human hantavirus infections in Switzerland, a seroepidemiological study was performed in 2009 on serum samples from 4,559 blood donors and 1,810 military personnel. Sera were screened using commercial Puumala IgG and hantavirus IgG enzyme-linked immunosorbent assays indicating a seroprevalence of 1% and 9%, respectively. Subsequently, the samples were analysed by immunofluorescence assay and immunoblot assay, showing a much lower prevalence, of 0.4% and 0.3%, respectively. Two of the serum samples achieved an 80% reduction in plaque-forming units in a neutralisation test. Statistical evaluation of questionnaires only identified an association of age (above 50 years) with hantavirus seropositivity when adjusted for sex (odds ratio: 2.36; 95% confidence interval: 1.10–5.05). This study provides baseline data (0.3–0.4%) for future monitoring of hantavirus seroprevalence in Switzerland and highlights the challenges in estimating the seroprevalence of these viruses in a country with very low endemicity.