Many chemicals in use end up in the aquatic environment. The toxicity of water samples can be tested with bioassays, but a metabolomic approach has the advantage that multiple end points can be measured simultaneously and the affected metabolic pathways can be revealed. A current challenge in metabolomics is the study of mixture effects. This study aims at investigating the toxicity of an environmental extract and its most abundant chemicals identified by target chemical analysis of >100 organic micropollutants and effect-directed analysis (EDA) using the acetylcholinesterase (AChE) bioassay and metabolomics. Surface water from an agricultural area was sampled with a large volume solid phase extraction (LVSPE) device using three cartridges containing neutral, anionic, and cationic sorbents able to trap several pollutants classes like pharmaceuticals, pesticides, PAHs, PCBs, and perfluorinated surfactants. Targeted chemical analysis and AChE bioassay were performed on the cartridge extracts. The extract of the neutral sorbent cartridge contained most of the targeted chemicals, mainly imidacloprid, thiacloprid, and pirimicarb, and was the most potent AChE inhibitor. Using an EDA approach, other AChE inhibiting candidates were identified in the neutral extract, such as carbendazim and esprocarb. Additionally, a metabolomics experiment on the central nervous system (CNS) of the freshwater snail Lymnaea stagnalis was conducted. The snails were exposed to the extract, the three most abundant chemicals individually, and a mixture of these. The extract disturbed more metabolic pathways than the three most abundant chemicals individually, indicating the contribution of other chemicals. Most pathways perturbed by the extract exposure overlapped with those related to exposure to neonicotinoids, like the polyamine metabolism involved in CNS injuries. Metabolomics for the straightforward comparison between a complex mixture and single compound toxicity is still challenging but, compared to traditional biotesting, is a promising tool due to its increased sensitivity.