Prothioconazole, a chiral triazole fungicide, is widely used to control Fusarium head blight (FHB) of wheat. Fusarium graminearum (F. graminearum), as the main pathogen of FHB, can produce many secondary metabolites including deoxynivalenol (DON), which threatens the health of humans and animals. However, some fungicides may stimulate F. graminearum to synthesize more DON under certain conditions. Until now, the fungicidal activity and enantioselective effect of prothioconazole enantiomers on DON production, transcriptome and metabolome of F. graminearum were unclear. The fungicidal activity of R-(−)-prothioconazole against F. graminearum was 9.12–17.73 times higher than that of S-(+)-prothioconazole under all conditions. Prothioconazole enantiomers can induce F. graminearum to synthesize more DON under 0.99 water activity (a_w) and 30 °C, especially R-(−)-prothioconazole. The expression levels of TRI6, TRI10 and TRI101 under R-(−)-prothioconazole treatment were significantly higher than those under S-(+)-prothioconazole treatment. Most genes in glycolysis, pyruvate metabolism, the target of rapamycin (TOR) signaling transduction pathway and the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling transduction pathway showed higher expression levels under R-(−)-prothioconazole treatment than uner S-(+)-prothioconazole treatment and the control. The peroxisome pathway displayed higher transcriptional activity under S-(+)-prothioconazole treatment compared with R-(−)-prothioconazole and the control. Based on metabolomic data, R-(−)-prothioconazole can significantly influence phenylalanine metabolism, and no significantly enriched pathway was found under S-(+)-prothioconazole treatment. These results are helpful to understand the risk of prothioconazole enantiomers on DON production of F. graminearum and uncover the relevant underlying mechanisms of prothioconazole enantiomers.