The structure, diversity, spatial and seasonal distributions of amphipod assemblages associated with intertidal Zostera (Zosterella) noltei meadows were studied around the Kneiss Islands (central Mediterranean Sea). This site represents a site of international interest in terms of its ornithological diversity (Important Bird Area, Ramsar Site and SPAMI). The amphipod fauna was sampled at 32 stations in spring 2014. A total of 6,482 individuals belonging to 78 species and 22 families were identified; among these taxa, Lysianassa ceratina is new for the Tunisian amphipod inventory, whereas 25 species are identified for the first time in the Gulf of Gabès. The dominant species are Cymadusa filosa, Microdeutopus gryllotalpa, Gammarus insensibilis, Dexamine spiniventris, Monocorophium insidiosum, Elasmopus rapax, Melita palmata and Leucothoe incisa. Four amphipod assemblages are identified using MDS analysis. The distribution of amphipod assemblages is linked to several environmental factors, such as sediment type, organic matter content and distance from the shore, which is itself associated with a clear inshore–offshore gradient. Seasonal variations of the amphipod assemblage patterns at eight stations, sampled between April 2014 and January 2015, show a maximum abundance and diversity during summer and autumn, with a minimum in winter. These seasonal fluctuations may be related to many factors such as variations of climatic factors (e.g. temperature, salinity), the growth and production rates of Zostera noltei meadows and seasonal patterns in the life cycle of dominant species.