This study investigated risk factors associated with positive serological status for Trypanosoma cruzi antibodies in 26 rural communities including 905 households, 2,156 humans, and 333 dogs in Lara State, Venezuela. Serology was performed with ELISA and MABA. Data were obtained from entomological, demographic, and clinical surveys. Risk factors were determined through binary logistic regression. Seroprevalence was 7.24% in humans and 6.9% in canines. Positive serological status was positively associated with the Rhodnius prolixus vector, age, maternal history of Chagas disease, tobacco chewing, presence of mammals and birds in the household, household disarray, mud-and-wattle outbuildings, and animal nests and burrows in the peridomicile, and negatively associated with tobacco and alcohol consumption, history of cancer, and storage deposits in the peridomile. In conclusion, Chagas disease in this rural area is an old phenomenon transmitted by R. prolixus or by the transplacental route, associated with socio-cultural habits related to poverty, sylvatic surroundings, and the host's medical history.