Bartonella spp. bacteria are significant human pathogens and the agents of bacterial zoonosis acquired from an animal companion. The aim of the study was to determine the seroprevalence of two of the most common Bartonella species B. henselae and B. quintana in various human populations. The studied groups included: alcoholics, intravenous drug users, veterinarians, cats' owners. Blood samples were collected and cultured on chocolate agar plates and in mouse fibroblasts L-929 cell line culture. The levels of Bartonella henselae IgM and IgG antibodies were determined by indirect immunofluorescence assay. Specific B. henselae IgG were detected in 48.3% of homeless alcoholics, in 45.0% veterinarians and in 53.3% cats' owners. The differences in the prevalence of B. henselae antibodies between the studied groups and a control group were statistically supported. No homeless intravenous drug users had specific B. henselae and B. quintana antibodies. High titers of B. quintana IgG antibodies were detected in two homeless alcoholics. Bartonella spp. was cultured on chocolate blood agar plates from blood samples from 2 alcoholics. The isolates were identified as B. henselae by PCR amplification of the riboflavin synthase gene (ribC). The results prove that B. henselae and B. quintana, emerging human pathogens, are present and widely distributed in Poland in such specific risk groups as: alcoholics, veterinarians and cats' owners.