Abstract: To achieve the comprehensive utilization of walnut, pyroligneous acid was prepared by pyrolyzing walnut shell. Pyroligneous acids of walnut shell collected at different temperature ranges, i. e., 90～150℃, 150～310℃ and 310～550℃ were obtained. Antimicrobial activities of each pyroligneous acid were tested by using different fungus or bacteria. Chemical constituents of the pyroligneous acid showing the strongest antimicrobial activities were analyzed by Gas Chromatography-Mass Spectrometry(GC-MS). The results showed that the temperature range of preparing pyroligneous acid by using walnut shell was 90～550℃; Yields of refined pyroligneous acid decreased along with the increase of pyrolysis temperature, while the densities and pH values increased, and the contents of organic acids decreased. All the pyroligneous acids collected at different temperature ranges showed antimicrobial activities, and significant differences existed with the control. 43 compounds were found by GC-MS analysis from the pyroligneous acid collected at the temperature range of 150～310℃, mainly organic acids, phenols, ketones and aldehydes. It was suggested that phenols, accounting for 80.70% of the total constituents detected, were the active components for antimicrobial activities.