A wide range of toxic organophosphorus pesticides and nerve agents is effectively hydrolyzed by the structurally related phosphotriesterase enzymes paraoxonase (PON1) from human plasma and diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris. Both enzymes have potential use as medical countermeasures and decontaminants. Enhanced enzymatic activity, stereochemical preference, and substrate variety are still the focus of ongoing research. Derivatives of pesticides and nerve agents bearing a fluorogenic leaving group were introduced for high-throughput screening of mutant libraries recently. We report the inhibitory potency of fluorogenic organophosphorus compounds with three different leaving groups [3-chloro-7-oxy-4-methylcoumarin, 7-oxy-4-methylcoumarin, 7-oxy-4-(trifluoromethyl)coumarin] toward human acetylcholinesterase (AChE) and report kinetic data for the enzymatic hydrolysis of these compounds by PON1 and DFPase. This is the first report of the hydrolysis of a substrate bearing a P-O bond to the leaving group by DFPase (its activity was believed to be restricted to cleavage of P-F and P-CN bonds). The reactivity of the enzymes toward the substrates is explained on the basis of structural reasoning and computational docking studies. We demonstrate that fluorogenic organophosphorus compounds can serve as valuable models for enzyme screening but also show that differences and limitations exist and have to be taken into account. The importance of using protein from human sources to obtain toxicological data for potential in vivo use is highlighted.