Pesticides are one of the most frequently anthropogenic xenobiotics detected in water. Among these, the organophosphorus pesticides (OPs) are very widely used in agriculture due to their broad spectrum of activity and their low price, but they also have high potent effects as neurotoxic compounds in non-target organisms. The aim of this study was to evaluate biomarkers acetylcholinesterase (AChE), butyrylcholinesterase (BChE), propionylcholinesterase (PChE) and carboxylesterase (CbE) in the representative Atlantic fish species Trachurus trachurus, Merluccius merluccius and Trisopterus luscus from "Rías Gallegas", a traditional Spanish fishing area. These esterase activities were evaluated in the brain, muscle and liver to determine the most adequate tissue to measure such enzymatic activities. The sensitivity of AChE and CbE activities from different tissues the widely used organophosphorus insecticide chlorpyrifos (CP), and its toxic metabolite (CP-oxon) was also tested. AChE activity was predominant in all tissues of the analysed species (particularly in brain constituting from 78.33%, 89.83% and 88.43% of total ChEs in Trachurus trachurus, Merluccius merluccius and Trisopterus luscus, respectively). Under in vitro exposure, esterases were shown to be highly sensitive to CP and especially to CP-oxon. Moreover, a similar effect observed on AChE and CbE activities could suggest that CbE activity might contribute efficiently against the toxic effects of CP, especially in muscle and the liver. The presence of BChE, PChE and upper CbE activities in muscle and the liver and their OP-sensibilities can be used to study their function in the pesticide biochemical detoxification pathways with a prominent role as a safeguarding mechanism against pesticide toxicity.