Skhira Bay located in the Gulf of Gabès, on the southeastern coast of Tunisia, is an important area in terms of its dense vegetation coverage, wide continental shelf, and fisheries resources. However, this area with a typically micro-tidal range is subject to intensive anthropogenic pressures: soft bottom trawling, chemical pollution from phosphoric acid production, and shipping activity. The present study is the first investigation of the structure of the benthic macrofauna on this part of the Tunisian coast. In April 2010, 28 stations were sampled along four transects from the phosphogypsum outfall on an inshore-offshore gradient. A total of 239 macrobenthos taxa, belonging to nine zoological groups and 140 families, were identified with a dominance of polychaetes (33.5%), crustaceans (29.4%), and mollusks (19.6%). Results show that the stations facing the phosphogypsum discharges are the most disturbed and characterized by a poorly diversified macrofauna. The macrofauna is dominated by carnivores, suspension feeders, and selective deposit feeders, and seems to be linked more to the availability of trophic resources than to disturbance. Four benthic assemblages are identified using Cluster and MDS analyses linked to edaphic factors, such as sediment structure, organic matter content, inshore/offshore gradient, and the proximity of the phosphogypsum outfall. The biotic indices (AMBI and BO2A) calculated from macrofauna data show that the ecological status of Skhira Bay varies overall from moderate to good. This study suggests initiating a long-term monitoring program to improve our understanding of the temporal changes of this ecosystem, to recommend the necessary conservation measures in this area of high-value natural heritage.