The mosquito-borne zoonotic nematode parasites, Dirofilaria immitis and Dirofilaria repens, primarily affect dogs. In recent years, their distribution has expanded due to various factors influencing vector-borne pathogens. This study aimed to investigate the comparative prevalence of infection in dogs and humans within a hyperenzootic region of Europe and to estimate the proportional relationship between infection prevalence in dogs and humans, in the concept of “One Health”. To this end, 604 blood samples from dogs and 625 serum samples from humans living in the Thrace region of northeastern Greece were collected. Dog samples were examined using Kott’s test for Dirofilaria spp. microfilariae and a commercial serological test for D. immitis antigen. Human sera were analysed by western blot for both parasites. The overall prevalence of infection in dogs was 177 (29.3%), with 173 (28.6%) testing positive for D. immitis and 7 (1.3%) for D. repens, including 6 (1%) cases of mixed infection. Specific IgG antibodies were detected in 42 (6.7%) human samples, with 24 (3.8%) positive for D. immitis and 18 (2.9%) for D. repens. The infection proportion in humans was 23.4% of the corresponding canine infection, indicating a high risk of human infection in this hyperenzootic region.