American trypanosomiasis is an important zoonotic disease which affects more than 15 million persons in America. In Mexico, Chagas’ disease is widely distributed in the country mostly in states with tropical weather conditions, including Yucatan. A cross-sectional study was performed on serum samples from 35 dogs and their owners (n = 75) from the south area of Merida city. Specific IgG antibodies against Trypanosoma cruzi (T. cruzi) using enzyme-linked immunosorbent assay, Immunofluorescence antibody test (IFI) and Western blot (WB) were detected. The overall percentage of seropositivity was 34% in dogs and 8% for sampled owners. Some owners brought samples of insect vectors found in their households, which were kept with BALB/c mice as a source of food and which were evaluated by polymerase chain reaction. All tested insects were positive to T. cruzi, and BALB/C mice were IFI and WB positive after 45 days in contact with these vectors. Further investigations showed that there is a high risk of infection with Chagas disease in dogs which spend the night outdoors (P < 0.05), with low body conditions score and older dogs. Risk factors identified to be associated to the infection in owners were gender and occupation (bricklayers, P < 0.05). The presence of seropositive dogs in houses where vectors are well-adapted represents a high risk for humans to become infected when bitten by a vector infected by a positive dog.