Background: Rickettsiosis are zoonotic diseases, caused by bacteria of the Rickettsia genus, which can be transmitted to humans through the bite or contact with feces of different arthropods, including ticks, lice, mites and fleas. Infection by these bacteria in humans is characterized for causing an acute febrile syndrome that can vary in the intensity depending on the bacteria species. Methods & Materials: A cross-sectional study was conducted between July and November 2017, in which 506 human samples were collected from healthy adult population from four municipalities of Cauca department, Colombia: Caloro, La Sierra, El Tambo and Santander de Quilichao. People who agreed to participate in the study that met the inclusion criteria and signed the informed consent previously endorsed by the Institutional Review Board from the Pontificia Universidad Javeriana. The samples were transported at −20 °C to the Special Bacteriology Laboratory where they were processed by indirect immunofluorescence for the detection of IgG antibodies against R. rickettsii Sheila Smith strain, R. typhi Wilmington and R. felis Pedreira strain. Titers higher than 1:64 were considered as positive. Results: 434/506 (85.8%) positive samples were obtained against R. rickettsii, 306/506 (60.4%) against R. typhi and 242/506 (47.8%) against R. felis, a cross-reactivity of 52.7% was presented between R. Rickettsii and R. typhi; 41.7% between R. rickettsii and R. felis and 32.60% between R. typhi and R. felis Conclusion: Seroprevalence of Rickettsia spp. infection in prioritized rural areas in the department of Cauca is high. The following results demonstrate that further studies need to be realized to determine if current cases of acute febrile syndrome are caused by Rickettsia spp and misdiagnosed with other diseases, thereby public health decisions can be taken for proper treatment and diagnosis of affected population.