A liquid chromatography with tandem mass spectrometry method was developed and applied for the investigation of penthiopyrad photolysation and hydrolysation in different aqueous solutions, and the potential enantioselectivity of this chiral fungicide was further assessed. Good linearity was obtained (R² > 0.99) for R-(−)- and S-(+)-penthiopyrad in different matrices and the respective limits of detection and quantitation of both enantiomers were 0.003 μg mL⁻¹ and 0.01 μg mL⁻¹. The most suitable illumination intensity for photolysing penthiopyrad was 400 W. Other factors such as pH, salinity and flavonoid also influenced penthiopyrad photolysation. In an acidic solution, the photolytic degradation rate of penthiopyrad enantiomers decreased due to their chemical structures and physicochemical characteristics. Different salinities affected penthiopyrad photolysation differently; acceleration occurred in solutions with salinities of 0.5% or 3.0% due to effective catalysis by photosensitive chloride radical anions, and when the salinity was 1.0% or 2.0%, the inhibitory effect of inorganic salt decreased the photolysation rate. The addition of different flavonoids could significantly inhibit the photolysation of penthiopyrad by decreasing degradation 5–597 times through photo-quenching effects. Under some conditions, the S-(+)-enantiomer was photolysed faster than its antipode, indicating that the photolysation of penthiopyrad was enantioselective. During the hydrolysation process, penthiopyrad degraded faster in the acidic solution than in the other two aqueous solutions with different pH levels, which was attributed to the hydrogen ion-induced catalytic reaction. In sterile/nonsterile natural water matrices, the hydrolysation rate of penthiopyrad enantiomers was also influenced by pH, in the degradation order of tap water (pH 7.1) > rain water (pH 7.3) > lake water (pH 8.8). Enantiopure enantiomers were hydrolysed more slowly than racemic enantiomers due to their mutual promotion effects. The configuration stability of penthiopyrad enantiomers was preserved during the degradation process. Similar enantioselectivities of the hydrolysation of penthiopyrad and preferential degradation of the S-(+)-enantiomer were observed in some aqueous solutions. The results could provide some data to more accurately demonstrate the enantioselective environmental fate and risk of penthiopyrad in aquatic environments.