Blood transfusions are indispensable in Veterinary Medicine, providing therapeutic support in cases of hematological disorders. Several pathogens can cause disease and/or exacerbate the condition of immunocompromised dogs or those requiring a transfusion. This study aimed to investigate the molecular occurrence of hemopathogens (Bartonella spp., Ehrlichia spp., Anaplasma spp., piroplasmids, and hemoplasmas) in blood donor and patient dogs using samples from a clinical veterinary laboratory in Brazil. One hundred blood samples were collected from each group. All dogs tested negative for Bartonella spp. in all performed assays. Among the 100 dogs from the clinical veterinary laboratory, 15% (95% CI: 9.3–23.3) tested positive for Ehrlichia spp., 6% (95% CI: 2.8–12.5) for Anaplasma spp., 3% (95% CI: 1.0–8.5) for Babesia spp., and 2% (95% CI: 0.6–7.0) for hemoplasmas. Blood donor dogs tested positive for hemoplasmas (5%) (95% CI: 2.2–11.2). Additional conventional and real-time PCR assays followed by sequencing confirmed the presence of Ehrlichia canis, Anaplasma platys, Babesia vogeli, ‘Candidatus Mycoplasma haematoparvum’, and Mycoplasma haemocanis. The molecular detection of E. canis, A. platys, ‘Ca. M. haematoparvum’, and M. haemocanis in dogs from midwestern Brazil reinforces the relevance of molecular tools in diagnosing hemopathogens. This is the first molecular detection of hemoplasmas in canine blood donors from Brazil. This finding indicates their silent circulation and highlights the importance of molecular screening to prevent the worsening of clinical conditions and the risk of turning recipients into new sources of infection.