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<i>Cymbopogon winterianus</i>, known as “citronella grass”, is an important aromatic and medicinal tropical herbaceous plant. The essential oil of <i>C. winterianus</i> (EOCw) is popularly used to play an important role in improving human health due to its potential as a bioactive component. The present study aimed to identify the components of the essential oil of <i>C. winterianus</i> and verify its leishmanicidal and trypanocidal potential, as well as the cytotoxicity in mammalian cells, in vitro. The EOCw had geraniol (42.13%), citronellal (17.31%), and citronellol (16.91%) as major constituents. The essential oil only exhibited significant cytotoxicity in mammalian fibroblasts at concentrations greater than 250 μg/mL, while regarding antipromastigote and antiepimastigote activities, they presented values considered clinically relevant, since both had LC₅₀ < 62.5 μg/mL. It can be concluded that this is a pioneer study on the potential of the essential oil of <i>C. winterianus</i> and its use against the parasites <i>T. cruzi</i> and <i>L. brasiliensis</i>, and its importance is also based on this fact. Additionally, according to the results, <i>C. winterianus</i> was effective in presenting values of clinical relevance and low toxicity and, therefore, an indicator of popular use.