Aquatic sediments act as a sink for pollutants that potentially impact on aquatic communities. However, spatial correlations between pollution, hydrology, catchment disturbance and other factors make it difficult to determine the impact of sediment pollution. Field-based microcosm experiments utilising aquatic macroinvertebrates are one approach to isolating the biological effects of sediment pollution on aquatic biota. A field-based microcosm experiment was used to assess the effects of sediment from 14 sites along the River Murray system, Australia. Aquatic ecosystem declines have been observed in this river, but few studies have investigated the quality of its sediments or their biological impact. Chironomidae (midge larvae) dominated the microcosm experiment and were useful as bioindicators of sediment quality. Community composition, high incidences of larval mouthpart deformities in Procladius paludicola and skewed sex ratios in Tanytarsus fuscithorax indicated sediments from irrigation districts were having a toxic effect, but only nutrients were detected at biologically relevant concentrations and these did not correlate with species responses. The present study showed that the biological endpoints used in the microcosm approach can elucidate sediment toxicity even in the absence of supporting sediment chemistry and could successfully be applied to examine changes in sediment quality along a river system.