In the present study, we aimed to perform a phytochemical investigation of the whole herb of Matricaria chamomilla L., a Uygur herbal medicine. A total of 18 phenolic compounds were isolated by silica gel and Sephadex LH-20 chromatography, together with preparative HPLC methods. By analysis of the MS and NMR spectroscopic data and comparison with those in literature, these 18 compounds were identified as apigenin (1), galangin (2), luteolin (3), kempherol (4), quercetin (5), hispidulin (6), 6-methoxykaempferol (7), eupafolin (8), 3-methylquercetin (9), ermanin (10), 5,7,4′-trihydroxy-3,6-dimethoxyflavonone (11),bracteoside (12), 7-O-(β-D-glucopyranosyl)-galactin (13), isochlorogenic acid B (14), isochlorogenic acid C (15), 4-hydroxybenzoic acid (16), 5-pentadecylbenzene-1,3-diol (17) and scopoletin (18). Among them, compounds 1–13 were identified as flavonoids. Compounds 2 and 6–17 were isolated from both the plant and the Matricaria genus for the first time.