Prothioconazole (PTC) is a broad-spectrum triazole fungicide with one asymmetric center and consists of two enantiomers,R-(−)-PTC andS-(+)-PTC. To address the concern of its environmental safety, the enantioselective toxic effects of PTC onScendesmus obliquus(S. obliquus) were investigated. PTC racemates (Rac-PTC) and enantiomers exhibited dose-dependent acute toxicity effects againstS. obliquusat a concentration from 1 to 10 mg·L−1. The 72 h-EC50 value ofRac-,R-(−)-, andS-(+)-PTC is 8.15, 16.53, and 7.85 mg·L−1, respectively. The growth ratios and photosynthetic pigment contents of theR-(−)-PTC treatment groups were higher than theRac- andS-(+)-PTC treatment groups. Both catalase (CAT) activities and esterase activities were inhibited in theRac- andS-(+)-PTC treatment groups at high concentrations of 5 and 10 mg·L−1, and the levels of malondialdehyde (MDA) were elevated, which exceeded the levels in algal cells for theR-(−)-PTC treatment groups. PTC could disrupt the cell morphology ofS. obliquusand induce cell membrane damage, following the order ofS-(+)-PTC ≈Rac-PTC >R-(−)-PTC. The enantioselective toxic effects of PTC onS. obliquusprovide essential information for its ecological risk assessment.