Biomarkers are used as tools to measure physiological, cellular, biochemical, and/or molecular changes, reflecting the level of disturbances and alterations caused by the exposure of organisms to chemical pollutants present in the environment. In the present study, a multi-biomarker approach was used to assess the effects of hydrocarbon contamination in the southern lagoon of Tunis (Tunisia), on clam Ruditapes decussatus. Sediments and clams were collected from four locations: three of them were located within the polluted lagoon (S1, S2 and S3) and another one was allocated in a clean site on the Mediterranean coast (SR). Total hydrocarbons were extracted from clam tissues and sediments. On the other hand, biomarkers of oxidative stress, neurotoxicity, and lipid peroxidation were measured. Hydrocarbon analysis revealed a high pollution state in the south lagoon of Tunis with a spatial variation. On the other side, our results showed activation of antioxidant enzymes in clams sampled from the polluted area as well as neurotoxicity. These results reveal the alteration of the health status in these organisms generated by the anthropogenic pollution in this area. Overall, the present work clearly showed that affected biomarkers could be useful tools for biomonitoring in the study area.