Information on the constituents of medicinal plants used in Sudanese system of medicine is very scarce. Hence, this study was set to investigate the flavonoids of Sudanese Acacia nilotica subspecies tomentoza which is a key species in indigenous medicine. A dihydrochalcone was isolated from roots using a combination of chromatographic techniques and its structure was partially elucidated on the basis of its spectral data (UV, ¹HNMR and MS). The antimicrobial activity of compound I was evaluated and significant activity was observed.