Helianthus tuberosus has been known to inhibit the growth of weeds and other plants sharing its habitat. This study was conducted to identify the allelochemicals of Helianthus tuberosus which were extracted with water and solvents. Aqueous extracts of leaf, stem, root, tuber and tuber peel of Helianthus tuberosus except tuber did not show significant differences in phytotoxicity to alfalfa seedlings. It was considered that Helianthus tuberosus contained fewer or less potential water-soluble substances that were toxic to alfalfa. Methanol extract of leaves of Helianthus tuberosus was sequentially partitioned in increasing polarity with n-hexane, ethylacetate and n-butanol. Each extract had a yield of 148, 12, 15.7 and 9.5g, respectively. Inhibitory effects on germination of alfalfa seeds treated with four fractions were not significantly different. But the significant reductions on hypocotyl length were observed for all the solvent extracts. Among the four fractions, the ethylacetate fraction showed the most significant inhibition effect on bioassay with alfalfa. Further separation of the active ethylacetate fraction by open column chromatography led to the 25 subfractions. In bioassay of each sub-fraction with alfalfa seeds, sub-fraction No. 13 showed the most inhibitory effect on seedling growth. 1 H NMR and gas chromatography-mass spectrometry analysis revealed that sub-fraction No. 13 was the mixture of straight-chain saturated fatty acids.