Pesticides are frequently applied to agricultural activities to improve harvest, in terms of yield and product quality. Useful tools for ecotoxicological studies of marine ecosystems are based on biomarker application on bioindicator key fish species. The main aim of the present study was to detect the potential presence of pesticides in a polluted coastal marine environment, namely Milazzo Gulf, situated in the north eastern coast of Sicily (Italy), by measuring the enzymatic activities of the ecotoxicological biomarkers acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in brain and blood samples of Chelon labrosus. Also, Marinello Reserve was selected as a reference site. The data showed a significant inhibition in AChE (81%) and BChE (71%) activities in fish from Milazzo Gulf in respect to those from the reference site. The esterase inhibition is primarily due to the presence of organophosphorus insecticides and carbamates that resulted, in Milazzo Gulf, higher in concentration in respect to the reference quality standard decree (D.M. 260, 2010). The results obtained in this study confirm the suspected presence of insecticides in waters and fish from Milazzo Gulf, which may lead to a considerable hazard to humans. This study confirms the relevant advantages of the biomarker approach on fish species in the ecotoxicological evaluation of marine environments.