The detection of pharmaceutical active compounds (PhACs) in the aquatic environment poses a serious concern for the wellness of biota due their ability to interfere with various biological targets (i.e., receptors, enzymes) causing alterations at different biological levels, including neurotransmission, antioxidant system and energy pathways. Among PhACs, the anti-inflammatory dexamethasone (DEX) results globally employed for the treatment of diseases, like arthritis, allergies, and asthma, but also as antiemetic interacting with the nervous system, particularly the serotonergic one. Therefore, the potential interference of DEX on the neurotransmission systems was evaluated on the non-target aquatic organism Mytilus galloprovincialis. The specimens were exposed to different DEX concentrations (4 ng/L-2000 ng/L), according to those recorded in the environment and wastewaters, considering three different times of sampling (3, 6, 12 days). A multi-biomarker approach (enzymatic, immunohistochemical and metabolomic analyses) was performed on mussel gills since their functions (i.e., gas exchange, filter feeding, osmoregulation) result regulated by serotoninergic, cholinergic, and dopaminergic neurotransmission systems. A severe neurotoxic effect in the serotoninergic and dopaminergic system caused by the tested doses of DEX was supported by a drop in the immunopositivity for serotonin (5-HT) and its receptor (5-HT₃R), as well as the enzyme tyrosine hydroxylase (TH), respectively. The effect on the cholinergic system revealed a similar trend with a reduction of acetylcholinesterase (AChE) immunopositivity coupled with an inhibition of its enzymatic activity, but with a partial recovery after 12 days of exposure as supported by a rise in the immunopositivity of choline acetyltransferase (ChAT) and a restoring of acetilcholine levels. The neurotransmission impairments, probably associated with alteration in the membrane integrity, seemed to be supported by changes in the levels of different osmolytes (i.e., taurine, betaine, homarine). Overall, this study confirms the environmental impact of PhACs on marine biota and the high evolutionary conservation of various drug biological targets.