In this study, non-targeted and targeted metabolomics/lipidomics studies based on UPLC-QTOF-MS and UPLC-MS/MS were carried out to clarify the effects of tebuconazole and its different enantiomers on lettuce metabolites and lipids. Slight enantioselective degradation of tebuconazole was observed and six degradation metabolites were tentatively identified. The endogenous metabolites involved in carbohydrate metabolism, amino acid metabolism, nucleic acid metabolism, phenylpropanoid and flavonoid metabolism, vitamins, and lipid metabolism were significantly affected with enantioselectivity by tebuconazole exposure. Nucleotide metabolism and nicotinic acid metabolic network were significantly activated by the stimulation of tebuconazole. Rac- and (-)-R-tebuconazole caused the down-regulation of soluble sugars and subsequent amino acids and organic acids. Overall, lettuce exposed to tebuconazole was shown to have a significant impact on plant metabolism and lipid metabolism, with notable stereoselectivity. The results showed stereoselective toxicity of tebuconazole and provided a better understanding of its metabolomic and lipidomic effects on lettuce.