The total flavones in the root of Zanthoxylum nitidum (Roxb.) DC was extracted with 70% ethanol and the content of the total flavones in it was determined to be 82.9 mg/g by spectrophotometry at wavelength 510 nm based on rutin as standard. The in vitro antimicrobial activities of the total flavones extract against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella sp, Aspergillus niger, Aspergillus flavus, Penicillium citrinum, Saccharomyces cerevisiae, Rhodotorula glutinis, Aspergillus oryzae were studied and the minimum inhibitory concentrations were measured to be 0.5, 1.0, 1.0, 1.0, 4.0, 1.0, 2.0, 2.0, 1.0, 1.0 mg/mL, respectively. The results indicate that the flavones in Zanthoxylum nitidum (Roxb.) DC possesses strong antimicrobial activity and potentially will be useful for the development of new chemotherapeutic agents against microbial infections.