Lake Faro (Sicily, Italy) is a natural confined brackish environment particularly subject to anthropogenic impact, resulting in a mixture of xenobiotic substances, i.e. heavy metals and polycyclic aromatic hydrocarbons (PAHs), and characterised by low hydrodynamics. In order to assess the water quality status of this pond, a multi-biomarker approach was applied on mussel Mytilus galloprovincialis (Lamarck, 1819) both inhabiting the lake and from a control site (Goro). Different biomarkers were investigated on mussel digestive glands and gills, including biomarkers of exposure (cytochrome P450 4, CYP4), neurotoxicity (acetylcholinesterase, AChE; choline acetyltransferase, ChAT), general stress (lysosomal membrane stability, LMS), and genotoxicity (micronucleus and comet assays). The results suggest significant responses related to the selected area. A statistically significant inhibition (P < 0.0001) of CYP4 in the digestive gland, and of AChE and ChAT in the gills, was found in specimens collected from Faro compared with the control. The lysosomal membrane stability of mussels from Lake Faro was lower than the reference site, while the DNA damages were significantly higher in mussels from the brackish area compared to the control. This study represents the first time the quality status of Lake Faro is assessed using a multi-biomarker approach on the mussel M. galloprovincialis, which proved to be suitable to identify the effects of environmental pollutants at molecular and cellular levels.