Thymelaea microphylla Coss. et Dur is an endemic desert species from Algeria belonging to Thymelaeaceae family. The dichloromethane-methanolic extract from aerial parts of T. microphylla was subjected to a silica gel column. Three flavonoids were isolated from the extract; isochamaejasmin (1), cis-tiliroside (2) and trans-tiliroside (3). The structures of the compounds were determined on the basis of the HPLC-TOF/MS, 1D and 2D NMR spectroscopic analysis. To the best of our knowledge, compound (1) was isolated from T. microphylla for the first time and there is no report in the literature. Moreover, in this study, antioxidant activity of the compound (3) was also evaluated.