A study was conducted in the spring of 2009, the winters of 2010 and 2013, and in the summer of 2012 at 13 stations in Boughrara Lagoon, Tunisia (southern Mediterranean). The country’s largest lagoon, it is considered to be an anthropogenically stressed area, though a major tourist centre—Djerba Island—is located along its northern shores. The lagoon bottoms were studied via analyses of grain size, surface sediment composition, total organic matter (TOM) and through the trophic and functional organisation of benthic macrofauna. The results indicate that the bottoms are composed of fine, medium or coarse sands and that sediment distribution is controlled by water movement. Estimation of TOM content revealed that the studied samples present both normal and imbalanced sediments. The structure and organisation of the lagoon’s benthic macrofauna are dominated by select deposit feeders and underwent significant changes during the period 2010–2013. Subjected for decades to increased pollution due to growing human activities in the surrounding area, Boughrara Lagoon now appears to be impacted by certain environmental/anthropogenic stressors, as indicated by the presence of pollution-tolerant bio-indicator species in the imbalanced area. The response of the lagoon ecosystem to changes in benthic sediment deposition provides a potential assessment tool for similar habitats elsewhere.