Malathion, diethyl 2-[(dimethoxyphosphorothioyl)sulfanyl]butanedioate, is an organophosphate used to control insect pests. Malathion contains a diethyl succinate moiety that is a known functional group susceptible to desymmetrizing enzymes such as esterases that selectively react with a single enantiomer. Purified rac-malathion was subjected to hydrolysis at the diethyl succinate moiety of malathion under various conditions using wild type pig liver esterase to form (S)-malathion (12 % ee) and ~ 3:2 mixture of α- and β-monoacids of (R)-malathion. Technical malathion could not be enriched due to the presence of esterase inhibitors. Further investigation of this resolution using a panel of six PLE isoenzymes also demonstrated formation of (S)-malathion, however, an improvement of up to 56 % ee was obtained.