Studies of the sediments of Hamilton Harbour, Lake Ontario, Canada, have shown variable degrees of pollution with a large number of organic and inorganic pollutants. Three areas in the Harbour – Windemere Basin, Cootes Paradise and Randle Reef – exhibited particularly high levels of contaminants, with concomitant impacts on benthic organisms. Sediment samples examined in this study were taken from a site in Randle Reef in Hamilton Harbour and a station (LE 23) in Lake Erie used as a reference sample. The samples were subjected to chemical fractionation to remove organic contaminants, with one fraction being used as total extract, and another aliquot being subjected to silica gel fractionation to give four fractions. Each fraction was chemically analysed and an aliquot used for bioassays following exchange with dimethylsulfoxide. Standard biotests were performed with Daphnia magna, Hyalella azteca, Pimephales promelas (fathead minnows) and Selenastrum capricornutum. Results from direct sediment contact bioassays indicated that sediment from Randle Reef is highly toxic, whereas the Lake Erie sample had no observable impacts. Total extract when added at 1% to the bioassays replicated the solid phase with acute toxicity for Randle Reef, and no effect on Lake Erie. For the silica gel fractionation, Fraction 1 (F1) showed similar toxicity as the total extract, even at low additions, whereas F2–4 gave variable and inconclusive results. F1 eluted with hexane contained the non-polar fractions, predominantly the polyaromatic hydrocarbons, and these compounds are the most likely causative agents for the highly acute toxicity observed in the sediment of Randle Reef.