| Summary: | This study aimed to investigate the chemical composition as well as the antibacterial, antiparasitic, and cytotoxic potentialities of the Brazilian <i>Chrysopogon zizanioides</i> root essential oil (CZ-EO) In addition, CZ-EO cytotoxicity to LLCMK<sub>2</sub> adherent epithelial cells was assessed. The major compounds identified in CZ-EO were khusimol (30.0 ± 0.3%), β-eudesmol (10.8 ± 0.3%), α-muurolene (6.0 ± 0.1%), and patchouli alcohol (5.6 ± 0.2%). CZ-EO displayed optimal antibacterial activity against <i>Prevotella nigrescens</i>, <i>Fusobacterium nucleatum</i>, <i>Prevotella melaninogenica</i>, and <i>Aggregatibacter actinomycetemcomitans</i>, with Minimum Inhibitory Concentration (MIC) values between 22 and 62.5 µg/mL and Minimum Bactericidal Concentration (MBC) values between 22 and 400 µg/mL. CZ-EO was highly active against the <i>L. amazonensis</i> promastigote and amastigote forms (IC<sub>50</sub> = 7.20 and 16.21 µg/mL, respectively) and the <i>T. cruzi</i> trypomastigote form (IC<sub>50</sub> = 11.2 µg/mL). Moreover, CZ-EO showed moderate cytotoxicity to LLCMK<sub>2</sub> cells, with CC<sub>50</sub> = 565.4 µg/mL. These results revealed an interesting in vitro selectivity of CZ-EO toward the <i>L. amazonensis</i> promastigote and amastigote forms (Selectivity Index, SI = 78.5 and 34.8, respectively) and the <i>T. cruzi</i> trypomastigote form (SI = 50.5) compared to LLCMK<sub>2</sub> cells. These results showed the promising potential of CZ-EO for developing new antimicrobial, antileishmanial, and antitrypanosomal drugs.
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