Background: Through literature retrieval, there has been no report on the research of the chemical components in Zanthoxylum avicennae (Lam.) DC. This paper extracted and determined the chemical components of the volatile oil in Z. avicennae, and at the same time, measured and evaluated the bioactivity of the volatile oil in Z. avicennae. Materials and Methods: We extract the volatile oil in Z. avicennae by steam distillation method, determined the chemical composition of the volatile oil by GC-MS coupling technique, and adopt the peak area normalization method to measured the relative percentage of each chemical composition in the volatile oil. Meanwhile, we use the Lethal-to-prawn larva bioactivity experiment to screen the cytotoxicity activities of the volatile oil in Z. avicennae, and using the slanting test-tube experiment to determine and evaluate its antibacterial activities in vitro for the eight kinds of plant pathogenic fungi in the volatile oil of the Z. avicennae. Results: The results show that 68 kinds of compounds are determined from the volatile oil of Z. avicennae. The determined part takes up 97.89% of the total peak area. The main ingredients in the volatile oil of Z. avicennae are sesquiterpenoids and monoterpene. The test results show that the volatile oil in Z. avicennae has strong antibacterial activities and cytotoxicity, with the strongest antibacterial activity against the Rhizoctonia solani AG1-1A. Conclusion: This research results will provide reference data for understanding the chemical composition of the volatile oil in the aromatic plant of Z. avicennae and its bioactivity, and for its further development and application.