Essential oils have been increasingly studied as preservatives for the food industries because of their biological properties. However, there are few studies on the antibacterial potential of the essential oil of the species Cantinoa carpinifolia Benth. Thus, the aim of this study was to extract and chemically characterize the essential oil from Cantinoa carpinifolia Benth. and evaluate its antibacterial potential against strains of Escherichia coli and Staphylococcus aureus. The essential oil was extracted by hydrodistillation and analyzed by gas chromatography using mass spectrometer and flame ionization detectors. The antibacterial potential against Escherichia coli and Staphylococcus aureus was evaluated by macrodilution, through cell viability tests, membrane permeability tests and electronic microscopy (SEM). The essential oil is composed principally of the α‐ thujone e β‐ thujone monoterpenes, and the minimum bactericidal and bacteriostatic concentrations of the essential oil were 6.25 and 0.39 μL mL−1 for E coli and S aureus, respectively. Bacterial strains were completely inactivated after 135 minutes (E coli) and 200 minutes (S aureus), respectively. Intracellular biological activity was observed for the essential oil because the harmful effects on both species of bacteria could be observed in the electromicrographs.