Estuaries are recognised globally as areas of high production, diversity and high economic value. Exploitation of the economic potential of estuaries and attendant infrastructural development plus expansion of human populations has resulted in degradation of estuaries worldwide. Many estuaries of northern Australia, in contrast, remain in good ecological condition. The estuarine fish fauna of the South Alligator River, Kakadu region of northern Australia was investigated to determine seasonal and spatial variation in composition. Beam trawls were used at 94 locations over the wet and dry seasons of 2012. In total, 81 taxa were collected, 26 of which had not previously been recorded in the river. Sciaenidae and Engraulidae dominated species richness and abundance. Samples were heterogeneous in terms of composition and abundance in a manner unrelated to any measured aspect of the habitat over which trawls occurred. Species richness was higher in the lower estuary than the upper estuary and more species were detected during the wet season. Turnover in assemblage structure between sampling occasions was associated with seasonal variation in discharge concordant with changes in salinity and potentially productivity. Compositional variation was largely ascribed to differential spawning phenology of adults and tolerance to fluctuating salinity. The Kakadu region supports a rich fish fauna moderately distinct from that observed elsewhere in the Northern Territory. Pronounced temporal turnover in assemblage structure and spatial variation at a variety of scales (i.e. within samples, and within and between rivers) collectively pose a challenge to management in light of ongoing and proposed development of the water resources of northern Australia, including its estuaries.