Cancer is a health problem affecting a large part of the world population. In this context, several research groups have investigated molecules with higher efficiency and lower side effects. The species Leonotis nepetifolia is a shrub belonging to the Lamiaceae family with cytotoxicity activity reported in literature. This paper describes the isolation of compounds hentriacontane, phytyl palmitate, stigmasteryl glucoside, 6,7-dimethoxy-5,3’,4’trihidroxyflavone, apigenin-7-O-glucoside and luteolin-7-O-glucoside from extracts and the chemical composition of essential oil from the leaves of Brazilian L. nepetifolia species in addition to its in vitro cytotoxic activities. All compounds were identified by a series of spectrometric and spectroscopic methods, mainly NMR (1D and 2D) and GC-MS, as well as by comparison with literature data. The cytotoxic activity of isolated compounds and essential oil was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, against tumor cell lines HCT-116 (human colon) and SF-295 (glioblastoma). The alcohol 1-octen-3-ol was the majoritary compound of the essential oil and the compounds hentriacontane, phytyl palmitate, stigmasteryl glucoside, apigenin-7-O-glucoside and luteolin-7-O-glucoside were describes for the first time in this species. All compounds tested and essential oil showed low cytotoxic activity for the cell lines tested, suggesting that other phytochemical studies should be conducted for the discovery of compounds responsible by cytotoxic activity of the species.