For many vector-borne organisms, dogs can be used as sentinels to estimate the risk of human infection. The objective of this study was to use dogs as sentinels for multiple vector-borne organisms in order to evaluate the potential for human infection with these agents in southeastern Brazil. Blood from 198 sick dogs with clinicopathological abnormalities consistent with tick-borne infections were selected at the São Paulo State University Veterinary Teaching Hospital in Botucatu and tested for DNA and/or antibodies against specific vector-borne pathogens. At least one organism was detected in 88% of the dogs, and Ehrlichia canis DNA was amplified from 78% of the blood samples. Bartonella spp. seroreactivity was found in 3.6%. Leishmania chagasi antibodies were detected in 1% of the dogs. There was no serological or polymerase chain reaction evidence of infection with Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia chaffeensis, Ehrlichia ewingii, and Rickettsia rickettsii. The full E. canis 16S rRNA gene sequence of one of the Brazilian strains obtained in this study was identical to the causative agent of human ehrlichiosis in Venezuela. Ehrlichia canis may pose a human health hazard and may be undiagnosed in southeastern Brazil, whereas exposure to the other organisms examined in this study is presumably infrequent.