The biochemical characterization of cholinesterases (ChE) from different teleost species has been a critical step in ensuring the proper use of ChE activity levels as biomarkers in environmental monitoring programs. In the present study, ChE from Oreochromis niloticus, Piaractus mesopotamicus, Leporinus macrocephalus, and Prochilodus lineatus was biochemically characterized by specific substrates and inhibitors. Moreover, muscle tissue ChE sensitivity to the organophosphate pesticide methyl-paraoxon was evaluated by determining the inhibition kinetic constants for its progressive irreversible inhibition by methyl-paraoxon as well as the 50% inhibitory concentration (IC50) for 30 min for each species. The present results indicate that acetylcholinesterase (AChE) must be present in the muscle from P. mesopotamicus, L. macrocephalus, and P. lineatus and that O. niloticus possesses an atypical cholinesterase or AChE and butyrylcholinesterase (BChE). Furthermore, there is a large difference regarding the sensitivity of these enzymes to methyl-paraoxon. The determined IC50 values for 30 min were 70 nM (O. niloticus), 258 nM (P. lineatus), 319 nM (L. macrocephalus), and 1578 nM (P. mesopotamicus). The results of the present study also indicate that the use of efficient methods for extracting these enzymes, their kinetic characterization, and determination of sensitivity differences between AChE and BChE to organophosphate compounds are essential for the determination of accurate ChE activity levels for environmental monitoring programs.