Enantioselectivity in environmental behavior and toxic effect of chiral pesticides has been received much attention. In this study, enantioselective bioactivity towards target organism Botrytis cinerea, acute toxicity and bioaccumulation in Eisenia fetida, and degradation in five kinds of soil under laboratory conditions regarding triazole fungicide tebuconazole were investigated. The results showed that fungicidal activity to Botrytis cinerea of R-(-)-tebuconazole was 44 times higher than S-(+)-tebuconazole with an order of R-(-)-tebuconazole > rac-tebuconazole > S-(+)-tebuconazole. No significant difference was found in acute toxicity of rac-, R-, and S-tebuconazole to E. fetida with 48-h EC50 of 10.78, 10.48, and 10.84 μg/cm2, respectively. Dissipation of tebuconazole in the five tested soils varied upon soil characteristics with half-life ranging from 32.2 to 216.6 days. Enantioselective and rapid dissipation of tebuconazole were observed in soils Hainan and Huajiachi, compared to the other soils. Enantioselective accumulation of tebuconazole in E. fetida was found with a preferential of S-(+)-tebuconazole although no significant difference in acute toxicity to E. fetida between rac-tebuconazole and enantiomers. The results indicated that S-(+)-tebuconazole with less fungicidal activity may be more likely to be accumulated in earthworm E. fetida. This research is helpful to better evaluate the environmental and ecological risk of tebuconazole on enantiomeric level.