Autotoxicity, defined as a deleterious allelopathic effect among individuals of the same plant species, is considered as one of the factors that contributes to replant failure. Tobacco, as an important cultured and economic crop over the world, has been often hampered by replant failure. In view of the seriousness of this problem, the allelochemicals of flue-cured tobacco and their allelopathic effects were investigated. The extracts of rhizosphere soil exhibited phytotoxic activities against Lactuca sativa and autotoxic activities against tobacco itself. Bioassay-guided fractionation of the extract led to the isolation of six compounds, the structures of which were elucidated by spectroscopic analysis. Among them, β-cembrenediol (1), di-n-hexyl phthalate (2), and bis­(2-propylheptyl) phthalate (3) showed observably phytotoxic activities against L. sativa seedlings and autotoxic activities on tobacco. The allelochemicals were then verified in the root zone soils of flue-cured tobacco by high-performance liquid chromatography (HPLC). These results provide new insights into the allelopathic mechanisms involved in the replant failure of flue-cured tobacco.