The chemical composition of the essential oil obtained from the roots of Bidens tripartita L. by hydrodistillation was investigated by gas chromatography-mass spectrometry. In total, 106 compounds were identified (97.1% of the total oil). The main components of the oil were α-pinene (15.0%), β-bisabolene (9.3%), p-cymene (6.0%), hexanal (5.7%), linalool (4.6%), p-cymene-9-ol (3.4%), β-elemene (2.6%), 2-pentylfuran (2.2%), and silphiperfol-6-ene (2.1%). The antibacterial and antifungal properties of the essential oil were evaluated against eight Gram-positive and 11 Gram-negative bacterial species and 10 fungal strains. The oil exhibited a strong antifungal activity.