Rickettsia rickettsii and Rickettsia sp. strain Atlantic rainforest, that is considered to represent a genetic variant of Rickettsia parkeri, are confirmed as being capable of infecting humans in Brazil. This study reports the detection and characterization, by PCR and nucleotide sequencing, of Rickettsia sp. strain Atlantic rain forest in Amblyomma ovale parasitizing a human, in ticks infesting dogs and in free-living ticks collected from the environment where the human infestation was recorded. The data contribute to our knowledge of infection rates in A. ovale with Rickettsia sp. strain Atlantic rainforest and identified an additional location in the state of São Paulo populated with ticks infected with this emerging pathogen.