Rehmannia is a medicinal plant in China. Autotoxicity has been reported to be one of the major problems hindering the consecutive monoculture of Rehmannia. However, potential autotoxins produced by the fibrous roots are less known. In this study, the autotoxicity of these fibrous roots was investigated. Four groups of autotoxic compounds from the aqueous extracts of the fibrous roots were isolated and characterized. The ethyl acetate extracts of these water-soluble compounds were further analyzed and separated into five fractions. Among them, the most autotoxic fraction (Fr 3) was subjected to GC/MS analysis, resulting in 32 identified compounds. Based on literature, nine compounds were selected for testing their autotoxic effects on radicle growth. Seven out of the nine compounds were phenolic, which significantly reduced radicle growth in a concentration-dependent manner. The other two were aliphatic compounds that showed a moderate inhibition effect at three concentrations. Concentration of these compounds in soil samples was determined by HPLC. Furthermore, the autotoxic compounds were also found in the top soil of the commercially cultivated Rehmannia fields. It appears that a close link exists between the autotoxic effects on the seedlings and the compounds extracted from fibrous roots of Rehmannia.