A large number of organophosphorous insecticides (OPs) are chiral compounds, and yet enantioselectivity in their environmental fate and effects is rarely addressed. In the present study, we isolated individual enantiomers of three OPs, profenofos, fonofos, and crotoxyphos, and evaluated enantioselectivity in their inhibition of acetylcholinesterase (AChE). Acetylcholinesterase inhibition by the enantiomers and racemates was determined in vivo in the aquatic invertebrate Daphnia magna and in Japanese medaka (Oryzias latipes) as well as in vitro with electric eel (Electrophorus electricus) and human recombinant AChEs. The overall results showed variable sensitivity between AChE enzymes from different species as well as variable magnitude of enantioselectivity in enzyme inhibition. The (—)-enantiomer of profenofos was 4.3- to 8.5-fold more inhibitory to AChE in vivo, whereas (—)-fonofos was 2.3- to 29-fold more potent than the corresponding (+)-enantiomer. The (+)-enantiomer of crotoxyphos was 1.1- to 11-fold more inhibitory to AChE than the (—)-enantiomer. In contrast, the in vitro results showed (+)-profenofos to be 2.6- to 71.8-fold more inhibitory than the (—)-enantiomer and (—)-crotoxyphos to be 1.6- to 1.9-fold more active than the (+)-enantiomer. The reversed direction of enantioselectivity observed between the in vivo and in vitro assays suggests enantioselectivity within toxicodynamic processes such as uptake, biotransformation, or elimination. Findings from the present study provide evidence of enantioselectivity in the AChE inhibition of chiral OPs in nontarget organisms and indicate the need to consider enantiomers individually when assessing environmental risk of these chiral pesticides.