Allelopathy, the negative effect on plants of chemicals released to the surroundings by a neighboring plant, is an important factor which contributes to the spread of some weeds in plant communities. In this field, Stellera chamaejasme L. (Thymelaeaceae) is one of the most toxic and ecologically-threatening weeds in some of the grasslands of north and west China. Bioassay-guided fractionation of root extracts of this plant led to the isolation of eight flavonoids 1–8, whose structures were elucidated by spectroscopic analysis. All compounds obtained, except 7-methoxylneochaejasmin A (4) and (+)-epiafzelechin (5), showed strong phytotoxic activity against Arabidopsis thaliana seedlings. Seedling growth was reduced by neochamaejasmin B (1), mesoneochamaejasmin A (2), chamaejasmenin C (3), genkwanol A (6), daphnodorin B (7) and dihydrodaphnodorin B (8) with IC₅₀ values of 6.9, 12.1, 43.2, 74.8, 7.1 and 27.3 μg/mL, respectively, and all of these compounds disrupted root development. Endogenous auxin levels at the root tips of the A. thaliana DR5::GUS transgenic line were largely reduced by compounds 1, 2 and 6–8, and were increased by compound 4. Moreover, the inhibition rate of A. thaliana auxin transport mutants pin2 and aux1–7 by compounds 1–8 were all lower than the wild type (Col-0). The influence of these compounds on endogenous auxin distribution is thus proposed as a critical factor for the phytotoxic effect. Compounds 1, 2, 4 and 8 were found in soils associated with S. chamaejasme, and these flavonoids also showed phytotoxicity to Clinelymus nutans L., an associated weed of S. chamaejasme. These results indicated that some phytotoxic compounds from roots of S. chamaejasme may be involved in the potential allelopathic behavior of this widespread weed.