Chengdu City (Sichuan Province) and Anshun City (Guizhou Province) are the regions that an invasive plant, Chenopodium ambrosioides L., invades very seriously in China. Chengdu City is located in the Sichuan Basin with a pleasant climate and superior hydrothermal conditions, but Anshun City is located in the Yunnan-Guizhou Plateau with karst land feature, thin soil, high altitude, and frequent drought. To explore the differences in the allelopathy of C. ambrosioides in different habitats, the C. ambrosioides plants grown in Chengdu and Anshun cities were selected as research objects because of their obvious differences in environmental characteristics. The volatile oils of the C. ambrosioides plants from Chengdu and Anshun were extracted by steam distillation, and their yields were 3.173 and 4.820 g/kg, respectively. The analysis results of gas chromatography-mass spectrometry (GC-MS) analysis showed that the C. ambrosioides volatile oils from Chengdu and Anshun contained 16 and 25 compounds, respectively, and α-terpinene and cymene were their common components. The concentrations of α-terpinene and cymene were determined based on their contents in the volatile oils from both cities. In this study, we used six receptor plant species, including Vicia faba L., Arachis hypogaea L., Allium tuberosum Rottl. ex Spreng., Fagopyrum esculentum Moench, Pisum sativum L., and Brassica campestris L., which are widely grown in the farmlands invaded by C. ambrosioides. These receptor plants were separately treated with eight treatments, including the treatments with the C. ambrosioides volatile oils from both cities and with α-terpinene, cymene, and α-terpinene+cymene extracted from the volatile oils from both cities. The activities and nuclear structures of leaf stoma guard cells were examined by using the epidermal strip method. The results showed that these cells had nuclear aberrations, including nuclear deformities, nuclear pyknosis, and nuclear translocations. Moreover, the stoma guard cell activities were significantly decreased (P < 0.05) or even completely absent under the eight treatments, and these effects became more significant with an increase in treatment concentrations, and the maximum mortality and nuclear aberration rate of stoma guard cells were 93.85% and 81.16%, respectively. Among the six crop plants tested, F. esculentum had the strongest allelopathic effect, followed by P. sativum, V. faba, A. tuberosum, A. hypogaea, and finally B. campestris, according to their sensitivity to C. ambrosioides volatile oil, α-terpinene, and cymene. In this study, the cytotoxicity of the volatile oil of C. ambrosioides from Anshun was greater than that from Chengdu, and the cytotoxicity of α-terpinene was greater than that of cymene. The higher the proportion of α-terpinene in the α-terpinene+cymene solutions, the stronger the cytotoxicity they exerted. These results suggested that the allelochemicals of C. ambrosioides damaged the stoma guard cell structures of receptor plants, and when C. ambrosioides invades the habitats with relatively poor environmental conditions, it releases increased amounts of allelopathic chemicals in its surroundings for its competitive advantages.