Rickettsia helvetica, a tick-borne member of the spotted fever group Rickettsiae, is a suspected pathogen in humans; however its role in animals is unknown. The aims of this study were to establish a R. helvetica-specific real-time TaqMan PCR assay and apply it to the analysis of tick vectors (potential exposure risk) and blood samples from Canidae and humans (prevalence of infection). The newly designed 23S rRNA gene assay for R. helvetica was more sensitive than a published citrate synthase (gltA) gene assay for several Rickettsiae. Blood samples from 884 dogs, 58 foxes, 214 human patients and 2,073 ticks (Ixodes spp.) collected either from vegetation or animals were analyzed. Although the maximal likelihood estimate of prevalence was 12% in unfed ticks and even 36% in ticks collected from animals, none of the 1,156 blood samples tested PCR-positive. Ticks from cats were more frequently PCR-positive than ticks from dogs. Sequencing of the 23S rRNA and/or gltA gene of 17 tick pools confirmed the presence of R. helvetica. Additionally, R. monacensis that has not been previously described in Switzerland was identified. In conclusion, R. helvetica was frequently detected in the tick population, but not in blood samples. Nevertheless, due to the broad host range of the Ixodes ticks and the high infestation rate with this agent (i.e. R. helvetica was 13-times more frequently in unfed ticks than the tick-borne encephalitis virus), many mammals may be exposed to R. helvetica. The described PCR assay represents an important tool to study this topic.