OBJECTIVE: Detection of Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, Rangelia vitalii, and Babesia canis vogeli using real-time PCR in dogs treated at the veterinary hospital of the Universidade Santo Amaro, located in the south zone, in the city of São Paulo, SP, Brazil. METHODS: DNA was extracted from 63 whole blood samples using the “PureLink Genomic DNA” extraction kit (Invitrogen®) according to the manufacturer's instructions and real-time PCR was performed to detect Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, Rangelia vitalii, and Babesia canis vogeli. RESULTS: In total, 23.8% (15/63) of the samples were positive by real-time PCR for at least one pathogen. Of these, 9.52% (6/63) were positive for Babesia canis vogeli and 14.2% (9/63) for Ehrlichia canis. No samples were positive for Rickettsia rickettsii, Rangelia vitalii, and Anaplasma platys. CONCLUSION: This study demonstrated, in an unprecedented way, the presence of B. canis vogeli and E. canis in dogs from fragmentation areas of the Atlantic Forest around the Guarapiranga Reservoir in the city of São Paulo, expanding knowledge on the dispersion of this agent in Brazil.