In the present study, a multi-biomarker approach was used to assess the biological effects of metal pollution in the southern lagoon of Tunis, on clam Ruditapes decussatus both in "hot" (in summer) and "cold" (in winter) seasons. Clams were collected in August 2015 and February 2016 from three sites of the lagoon and from Louza considered a reference site. The concentrations of five trace metals (cadmium, copper, iron, lead, and zinc) in the soft tissues of R. decussatus were evaluated at the sampling sites. A core of biomarkers indicative of (a) neurotoxicity (acetylcholinesterase, AChE); (b) biotransformation (glutathione S-transferase, GST); (c) oxidative stress (catalase, CAT; total glutathione peroxidase, T-GPx; total glutathione peroxidase, T-GPx; selenium-dependent glutathione peroxidase, Se-GPx; glutathione reductase, GR; superoxide dismutase, SOD) (d) lipid peroxidation (malondialdhyde, MDA level), and (e) apoptotic process (caspase 3-like, CSP3) was selected for measurements of environmental effects on the populations of clams collected from the different sampling sites. The results of metal bioaccumulation in soft tissues of Ruditapes decussatus revealed a high pollution in the South Lagoon of Tunis with spatial variation and relatively high levels at the navigation channel. Anthropogenic pollutants in the lagoon led to the activation of antioxidant defense and biotransformation enzymes to oxidative damage of the membrane and activation of apoptosis, and revealed neurotoxicity. Among this core of biomarkers, the antioxidants enzymes (CAT, SOD, GR, and GPx) were very sensitive, allowing the discrimination among sites and pointing to the navigation channel as the most impacted site in the southern lagoon of Tunis. Moreover, a significant effect of season was recorded on biomarkers responses (e.g., CAT, GR, SOD, AChE, and CSP3 activities and MDA levels) with higher levels in winter than in summer, probably influenced by the reproductive stage and food availability. Finally, the measurement of the selected core of biomarkers in the whole soft tissues of clams was considered as an integrated indicator of environmental stress. Moreover, R. decussatus proved to be a remarkable sentinel species capable to establish a reliable diagnosis of the health status of the marine environment in different areas of the southern lagoon of Tunis, both in "hot" and "cold" seasons.