Ecotoxicological risk assessment for pesticides was performed on a coastal lagoon ecosystem (Orbetello lagoon, central Italy) according to the procedure described in the European technical guidance document (TGD) on risk assessment for new and existing substances. The procedure was applied to individual chemicals and to a mixture of chemicals with the same toxicological mode of action. Biomarker studies, measuring acetylcholinesterase (AchE) activity inhibition, were conducted on Zosterisessor ophiocephalus to compare the results of theoretical risk assessment with experimental measurements of ecotoxicological stress. Value and limitations of the risk assessment procedure and its application to site-specific environmental conditions are discussed. A tool crop risk index (CRI) for assessing the overall impact of pesticide use on a crop is proposed.