Rondonia state reports more spotted fever (SF) cases than any other state in the Brazilian Amazon. Ariquemes is the only municipality that has reported SF cases in this state, but no information has been obtained about the tick and Rickettsia spp. responsible. This study aimed to describe the free-living tick fauna and search for rickettsial DNA in ticks collected from a region where one specific infection was most likely acquired. Ticks were collected at four sites in Ariquemes municipality, and at one site in both Alto Paraíso and Rio Crespo municipalities. A total of 1082 ticks and eight larvae clusters were collected, comprising 12 species from the genus Amblyomma. The most abundant tick species found in this study were Amblyomma oblongoguttatum, A. scalpturatum and A. naponense. All ticks were tested for the presence of citrate synthase (gltA) and the outer membrane protein A (ompA) gene fragments to detect any Rickettsia. No rickettsiae amplification was observed; however, we did record the presence of A. ovale in the region, including at the probable site infection for a human spotted fever case. This tick is a known vector of Rickettsia parkeri Atlantic rainforest strain in Brazil. These results reinforce the need for further studies of ticks and tick-borne agents in order to improve our knowledge of tick-borne diseases in the Amazon at large.