Abstract Split-beam hydroacoustics revealed large numbers of small fish in the lower reaches of the Murray River in autumn. At slow-flow sites, acoustically estimated fish biomass could reach 102 kg/ha in mid channel, which compares with the biomass level of planktivorous fish in eutrophic lakes. These results were confirmed by direct catches with a push net of Australian smelt ( Retropinna semoni) and juvenile bony herring ( Nematalosa erebi) ranging in length from 18 to 150 mm. Fish densities in the river channel doubled at night compared to the day. The density and total biomass estimates from net catches were strongly correlated with the acoustic data. However, the catch estimates were only 50–60% of the acoustic measurements, which is explained by the limited netting efficiency. The two methods produced similar estimates of fish mean weights. These results suggest that the density and biomass of small fish in open water habitats of the river can be reliably determined with acoustics. The high biomass of planktivorous fish and the diverse zooplankton community found in the same habitat suggest that the fish probably exploit an advantageous ecological niche in the main channel of the Lower Murray. The high biomass of small fish in this reach of the river, which has previously been underestimated, provides a large potential food source for native predators.