The antifungal activity of essential oil (EO) from the Brazilian epazote (Chenopodium ambrosioides L.) was evaluated by the poison food assay at concentrations of 0.3%, 0.1%, and 0.05% with eight postharvest deteriorating fungi (Aspergillus flavus, Aspergillus glaucus, Aspergillus niger, Aspergillus ochraceous, Colletotrichum gloesporioides, Colletotrichum musae, Fusarium oxysporum, and Fusarium semitectum). EO components were tentatively identified by Kováts retention indices (RIs) using gas chromatography and gas chromatography combined with mass spectrometry (GC-MS). Growth of all fungi was completely inhibited at 0.3% concentration, and by 90% to 100% at 0.1% concentration. The following 13 tentatively identified compounds (relative percent) accounted for 90.4% of the total volatile oil: alpha-terpinene (0.9), p-cymene (2.0), benzyl alcohol (0.3), p-cresol (0.3), p-mentha-1,3,8-triene (0.2), p-cimen-8-ol (0.6), alpha-terpineol (0.5), (Z)-ascaridole (61.4), piperitone (0.9), carvacrol (3.9), (E)-ascaridole (18.6), (E)-piperitol acetate (0.5), and (Z)-carvyl acetate (0.3). Autobiographic thin layer chromatography of the EO to separate the principal fungitoxic fraction yielded only one fraction that completely inhibited the growth of all test fungi at a concentration of 0.1%. This fraction was characterized by RIs and GC-MS presenting a composition (%) of p-cymene (25.4), (Z)-ascaridole (44.4), and (E)-ascaridole (30.2). The results suggest ascaridoles were the principal fungitoxic components of the EO.