Abstract Haematopota pluvialis is a widely distributed hematophagic insect occurring across Eurasia. This horse fly may be a highly efficient mechanical vector of pathogens, including viruses, bacteria, and protozoa. Furthermore, its painful bites can cause local skin lesions and systemic symptoms. The aim of this study was to determine human exposure to H. pluvialis attacks in various types of open space habitats in Eastern Poland and to perform molecular screening of these tabanids for the presence of hematopathogens: Bartonella spp. and Anaplasma phagocytophilum. Specimens of H. pluvialis were collected at three distinct sites in Eastern Poland. The presence of Bartonella spp. and A. phagocytophilum was investigated using PCR-based methods. In total, 141 H. pluvialis females were analyzed. The molecular analysis of the rpoB gene fragment yielded one new haplotype of Bartonella sp. in 0.7% (1) of all studied samples, which may hypothetically exhibit zoonotic potential. Anaplasma phagocytophilum was not detected in the studied material. Moreover, a high level of human and animals exposure to horse fly bites was noted in the studied areas of Eastern Poland. The present results highlight the need for further targeted research on H. pluvialis to quantify pathogen prevalence, transmission efficiencies, and conditions facilitating pathogen transmission in natural settings. Keywords: Haematopota pluvialis; Bartonella; zoonotic pathogens; Tabanidae bites; Haematopota bites; Poland; Europe