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Enantiomers of chiral fungicides usually display different toxic effects on nontarget organisms in the surrounding environment, although there are rare reports on the enantioselective toxicity of metconazole (MEZ) to aquatic organisms, such as Microcystis flos-aquae (M. flos-aquae). To explore the enantioselective toxicity of MEZ in algae, the impact of various concentrations (0.001, 0.003, 0.01, 0.03 and 0.1 mg/L) of MEZ on M. flos-aquae over 8 days was investigated. Significant differences were observed between the four enantiomers in chlorophyll a (Chl a) contents, carotenoids, photochemical efficiency (Fv/Fm), rapid light-response curves (RLCs), utilization efficiency of light energy (α) and protein contents during treatment time. MEZ can enantioselectively stimulate the chlorophyll fluorescence parameters (RLCs, Fv/Fm and α) and carotenoid and Chl a contents of M. flos-aquae, especially at low concentrations (0.001 or 0.003 mg/L). At high concentrations of 0.03 or 0.1 mg/L, the chlorophyll fluorescence parameters (RLCs, Fv/Fm and α), protein and Chl a contents of M. flos-aquae exposed to cis-enantiomers were lower than those of M. flos-aquae exposed to trans-enantiomers. These observations indicated that the enantiomers of MEZ pose different toxicities to M. flos-aquae, with the cis-enantiomers more toxic than the trans-enantiomers. These results are beneficial for understanding the enantioselective effects of MEZ enantiomers on nontarget organisms and helpful for evaluating their eco-environment risk.