A wide variety of mammals are involved in the sylvatic cycle of Trypanosoma cruzi, the causative agent of Chagas disease. In many areas in Latin America where T. cruzi is endemic, this cycle is poorly known, and its main reservoirs have not been identified. In this study we analyzed T. cruzi infection in bats and other small mammals from an Ecological Reserve in southeastern Mexico. From January through March 2021, we captured wild individuals to extract cardiac and peripheral blood, and infection was detected by PCR of the mini-exon gene. In bats, the prevalence of infection was 16.36%, while in small mammals the prevalence was 28.57%. All of the samples that were positive for T. cruzi were identified as the TCI genotype. Our findings suggest that this zone, situated at the periphery of urban zones might have epidemiological relevance in the sylvatic cycle of T. cruzi and needs to be monitored. The infection of bats in this area is particularly concerning since the flight pattern of this populations overlaps with human settlements. Despite being subject to conservation protections, there continue to be anthropogenic actions that disturb the study area, which could exacerbate risks to public health.