There is evidence that the constituents of essential oils from plants of the genus Croton, such as Croton argyrophyllus Kunth, can be used for anti-inflammatory, antiproliferative, antidiabetic and antitumor purposes. As this is a genus of plants widely used among the communities of the semiarid region of Brazil, the present study aimed to identify the chemical composition and analyze the cytotoxicity of the essential oil from C. argyrophyllus Kunth. The essential oil was obtained through hydrodistillation from fresh leaves (0.33% yield) and analyzed by gas chromatography–mass spectrometry/flame ionization detector (GC–FID). The cytotoxic concentration of the essential oil of C. argyrophyllus by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test against the J774 macrophage was found to be 50 μg/mL. Twenty-five compounds were identified, representing 97.37% of the total oil, and the chemical profile was based on sesquiterpenes, with bicyclogermacrene (27.78%) its main constituent. The results demonstrate that the essential oil from leaves of C. argyrophyllus is cytotoxic at 50 and 100 μg/mL.