The essential oil from Phytolacca dodecandra, a traditional herb of Ethiopia, has been studied, including the chemical composition and antimicrobial activity. The difference between four P. dodecandra samples (P-1–P-4), which differed in gender or location, has also been analyzed. The essential oils were obtained by steam distillation, while the aromas were extracted by head space solid-phase microextraction (HS-SPME) and both were analyzed by gas chromatography- mass spectrometry (GC-MS). The oils’ antimicrobial activities were evaluated by the microdilution method against Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans. Ninety one components, representing 88.37 to 94.01% of the aromas, were identified. The compositions of the aromas of four samples are mainly dominated by aldehydes and ketones: 2-nonanone (1.80–30.80%), benzaldehyde (4.99–25.99%), and sulcatone (2.34–5.87%). Sixty components representing 64.61 to 69.64% of the oils were identified, and phytone (3.04–21.23%), phytol (4.11–26.29%) and palmitic acid (1.49–23.87%) are the major compounds. No obvious antimicrobial activity was observed for all the four essential oils.