Ehrlichia canis, Anaplasma platys, and Babesia vogeli are frequently detected in the veterinary routine with varied pathogenesis that can lead to death, mainly in co-infections. Although canine hemotropic mycoplasmas are considered of low clinical importance, they have recently gained prominence in molecular research. The present study aimed to analyze two hospital populations of dogs, randomly selected from the year 2013 and 2015, from the municipalities of Toledo (n=68) and Cascavel (n=107). Direct examination of blood smears, serology, and PCR were used to detect the presence of E. canis, A. platys, B. vogeli and Mycoplasma sp. Direct blood smear examination was negative for the investigated agents in all samples. Serum immunochromatography (SensPERT ™, VetAll Laboratories, Korea) in 175 animals showed that only 4% (n = 7) had anti-E. canis antibodies, while 60% (n = 105) were positive for Anaplasma sp. All PCR samples were negative for E. canis, while 18.28% (n = 32) were positive for A. platys, 3.42% (n = 6) positive for Babesia vogeli, and 7.41% (n = 13) positive for Mycoplasma sp. The study revealed the presence of A. platys in Toledo and Cascavel by PCR, which accentuates the need to monitor vector populations and usual hosts and to evaluate the potential risk of infection in humans.