The essential oil obtained by steam distillation of dried aerial parts of Ambrosia trifida L. from Northeast China was analyzed by GC and GC-MS. The essential oil yield based on dried plant material was 0.12% and thirty-five compounds (corresponding to 86.7% of the total weight) were identified. The main components were: bornyl acetate (15.5%), borneol (8.5%), caryophyllene oxide (8.3%), alpha-pinene (8.0%), germacrene D (6.3%), beta-caryophyllene (4.6%), trans-carveol (2.9%), beta-myrcene (2.6%), camphor (2.4%) and limonene (3.2%). A. trifida essential oil demonstrated bactericidal and fungicidal activity against six bacterial strains and two fungal strains, using the agar diffusion method.