Spotted fever illness caused by the tick-borne pathogen Rickettsia parkeri has emerged in the Pampa biome in southern Brazil, where the tick Amblyomma tigrinum is implicated as the main vector. Because domestic dogs are commonly parasitized by A. tigrinum, this canid is also a suitable sentinel for R. parkeri-associated spotted fever. Herein, we investigate rickettsial infection in ticks, domestic dogs and small mammals in a natural reserve of the Pampa biome in southern Brazil. The ticks A. tigrinum, Amblyomma aureolatum and Rhipicephalus sanguineus were collected from dogs. Molecular analyses of ticks did not detect R. parkeri; however, at least 34% (21/61) of the A. tigrinum ticks were infected by the non-pathogenic agent ‘Candidatus Rickettsia andeanae’. Serological analyses revealed that only 14% and 3% of 36 dogs and 34 small mammals, respectively, were exposed to rickettsial antigens. These results indicate that the study area is not endemic for R. parkeri rickettsiosis. We tabulated 10 studies that reported rickettsial infection in A. tigrinum populations from South America. There was a strong negative correlation between the infection rates by R. parkeri and ‘Candidatus R. andeanae’ in A. tigrinum populations. We propose that high infection rates by ‘Candidatus R. andeanae’ might promote the exclusion of R. parkeri from A. tigrinum populations. The mechanisms for such exclusion are yet to be elucidated.