Leishmaniases are a group of diseases caused by more than 20 species of the trypanosomatid parasite Leishmania, which affects 12 million people in 98 countries, and one billion are at risk of infection. There are different clinical forms, of which visceral leishmaniasis (VL) is the most aggressive. An increase in VL cases has been observed in the southern cone of Latin America. In Uruguay, it was detected in dogs for the first time in 2015, in the countryside. Therefore, there is an imminent need to develop new therapies for the treatment of leishmaniasis. With this objective, we evaluated the activity of a series of 50 compounds in vitro against Leishmania infantum, Leishmania amazonensis, and Trypanosoma brucei. Seventeen compounds from our chemical collection with good anti-trypanosomatid activity were found, with IC₅₀ values in the range of 90 nM - 25 µM and selectivity greater than 10 concerning IC₅₀ in murine macrophages. The selectivity of these active compounds was better than that of the reference drugs: glucantime and miltefosine. Also, using the Pathogen box chemical collection, we discovered five hits to be used in drug design and development. In vivo toxicity studies and proof of concept were also carried out in the murine model of the cutaneous form of the disease. Three of the evaluated compounds were active over the in vivo murine model of infection with 100% survival of infected animals. These results encourage us to continue the development of these molecules as anti-leishmanicidal agents.