Trypanosoma and Leishmania infections affect wild and domestic animals and human populations. The growing process of deforestation and urbanization of Atlantic Rainforest areas has given rise to introduction of humans and domestic animals to the sylvatic cycles of Trypanosoma and Leishmania species. Serological, parasitological, and molecular surveys among wild and domestic animals in the Corrego do Veado Biological Reserve, which is an Atlantic Rainforest fragment in the state of Espírito Santo, southeastern Brazil, were evaluated. In total, 154 wild animals of 25 species and 67 domestic animals (47 dogs and 20 horses) were sampled. All the domestic animals were serologically negative for anti-Leishmania infantum chagasi antibodies and negative in parasitological approaches. Only the Order Chiroptera presented positive blood cultures and cryopreserved isolates. The phylogenetic trees based on SSU rDNA and gGAPDH genes confirmed the occurrence of Trypanosoma dionisii and provided the first record of Trypanosoma cruzi marinkellei in southeastern Brazil. The studies conducted in Atlantic Rainforest remaining trees provide the knowledge of parasite diversity or detect parasites that can accelerate the loss of hosts diversity.