This study was aimed to investigate the major flavonoid of Ziziphus abyssinica root and to screen the antimicrobial activity of root ethanol extract. The flavonoids were extracted with aqueous ethanol and the crude extract was purified by paper chromatography where a flavonoid (compound I) was isolated. The structure of this compound has been partially characterized by some spectral tools (UV and ¹HNMR). In the antimicrobial assay, the root ethanol extract showed moderate activity against Staphylococcus aureus and the yeast Candida albicans. The extract also exhibited partial activity against other test organisms(. Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli).