Sediment contamination may occur from various anthropogenic activities, such as mining-, agricultural- and industrial practices. Many of the contaminants arising from these activities enter the aquatic system and precipitate from the surrounding water, becoming bound to sediment particles. These bound contaminants may reach concentrations higher than in the overlying water. Although water quality may be acceptable, an aquatic system may still be at risk if the contaminated sediment were to be disturbed through flooding, bioturbation or changes in the water chemistry. These contaminants may then desorb into the water column and prove detrimental to life forms in contact and dependent on that water source. Sediment quality monitoring has been a widespread international initiative and has led to the development of sediment toxicity assessment methods. This study focused on sediment bioassays, namely, Phytotoxkit, Ostracodtoxkit F and the Diptera bioassay, in assessing sediment quality of the Tweelopiespruit-Rietspruit-Bloubankspruit river system in Gauteng, South Africa. This river is known to have been impacted by acid mine drainage (AMD) since late August, 2002. Exposure of river sediment from 7 sampling sites to these bioassays provided an eco-toxicological estimation of the acute toxicity and chronic toxicity emanating from the contaminated sediments. Physico-chemical analyses revealed higher levels of sediment contamination closer to the mine. The bioassays displayed a similar trend with greater sensitivities to sediments closer to the mine and lower sensitivities to the less contaminated sites further downstream. AMD was therefore the main driver for sediment contamination. Whilst not all contaminants were bioavailable, statistical analysis showed that there were significant correlations between the elevated contaminant concentrations closer to the mine and bioassay responses.Keywords: acid mine drainage, sediment toxicity, bioassay, Ostracodtoxkit F, Phytotoxkit, Diptera bioassay