Periophthalmodon septemradiatus (Hamilton, 1822) is a mudskipper of the Mekong Delta that can be found along estuaries and lower reaches of rivers. In the present study, we determined diet and feeding ecology of this species by analyzing the contents within the stomachs of 1360 fish samples collected from August 2017 to July 2018. Data analysis suggested that P. septemradiatus is a carnivorous fish. We found six main food item categories: small fishes, prawns (Acetes spp.), crabs (Uca spp.), mollusks, ants (Dolichoderus sp.), and detritus. Both males and females at different sizes, seasons, and habitats ingest primarily Dolichoderus sp., secondarily detritus, and rarely other prey. Diet composition was similar between sexes but varied according to fish size, season, and habitat. Dolichoderus sp. and detritus regulate the spatial variation of food composition. Our findings contribute to future artificial cultivation for conservation.