The Naf River estuary is one of the most productive ecological ecosystems in Bangladesh providing an important feeding area for fishes and other aquatic animals. However, detailed information on macrobenthic communities is rarely available in this area. Our study focused on the seasonal and spatial variability of macrobenthic community structure with their biomass and functional guilds. In total, forty-seven taxa were identified under nine major groups and seven functional feeding guilds. Among macrobenthic taxa, Polychaeta was dominant in both seasons and all over the estuary contributing 60% of total benthos. The mean abundance of macrobenthos was higher during the pre-monsoon (2 972±1 994 inds./m2) than the monsoon (1 572±361 inds./m2) being maximum at mid-estuary region and minimum at upper and lower estuary regions. Of the diversity indices, density (P=0.01, P=0.003), the number of taxa (P=0.000 3, P=0.000 9) and Margalef’s Index (P=0.000 4, P=0.001) of macrobenthos were significantly different among stations and seasons. Shannon-Weiner index (P=0.009, P=0.12) and Pielou’s evenness index (P=0.03, P=0.14) had significant differences among stations but not among seasons. Cluster analysis suggested that distribution of macrofauna is strongly influenced by seasonality. Among the feeding types, carnivorous were found to be dominant at the upper and mid-estuarine regions, and herbivorous were at lower estuarine regions. Carnivorous species were dominant in both seasons. Omnivores have shown to be the most abundant feeding type in terms of biomass though herbivores were highest during monsoon.