Pterodon emarginatus Vogel, Fabaceae, is a native aromatic tree distributed by central region of Brazil. Hydroalcoholic infusions of the seeds are used in folk medicine for their anti-rheumatic and anti-inflammatory properties. The objective of this work was identified the chemical components and verify the cytotoxic effect of the essential oil (EO) from P. emarginatus seeds. Thus, the EO of P. emarginatus seeds was analyzed by GC/MS analysis followed by brine shrimp lethality test and cytotoxic activity against tumor cell lines and human peripheral mononuclear blood cells (PBMC). The cancer cell lines tested were C 6 (rat glioma), MeWo (human melanoma), CT 26 .WT (mouse colon carcinoma), MDA (human breast cancer), A 549 (human lung carcinoma), B 16 -F 1 (mouse melanoma), CHO-K1 (hamster ovary cell) and BHK-21 (hamster kidney fibroblast). Eleven compounds were identified by GC and CG/MS analyses. The main compounds with concentrations higher than 5% were β-elemene (15.3%), trans-caryophyllene (35.9%), α-humulene (6.8%), germacrene-D (9.8%), bicyclo germacrene (5.5%) and spathulenol (5.9%). The EO of P. emarginatus seeds showed toxicity to Artemia salina (LC50 1.63 μg/mL) and was active against all the cell lines tested. The potent cytotoxic activity had IC50 values ranging from 24.9 to 47 μg/mL. However, EO (1-100 µg/mL) had less cytotoxicity in PBMCs isolated from a healthy subject. In summary, the present study showed the potential antiproliferative of the EO of P. emarginatus seeds.