Chagas Disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, affecting 6–8 million people, mainly in Latin America. The medical treatment is based on two compounds, benznidazole and nifurtimox, with limited effectiveness and that produce severe side effects; consequently, there is an urgent need to develop new, safe, and effective drugs. Amphotericin B is the most potent antimycotic known to date. A21 is a derivative of this compound with the property of binding to ergosterol present in cell membranes of some organisms. In the search for a new therapeutic drug against T. cruzi, the objective of this work was to study the in vitro and in vivo effects of A21 derivative on T. cruzi. Our results show that the A21 increased the reactive oxygen species and reduced the mitochondrial membrane potential, affecting the morphology, metabolism, and cell membrane permeability of T. cruzi in vitro. Even more important was finding that in an in vivo murine model of infection, A21 in combination with benznidazole was able to reduce blood parasitemia, diminish the immune inflammatory infiltrate in skeletal muscle and rescue all the mice from death due to a virulent T. cruzi strain.