Bitumen mined in the oil sands region of Northern Alberta, Canada, is diluted with natural gas condensates to form dilbit, which is transported through pipelines. Sections of these pipelines come close to freshwater ecosystems. If dilbit is spilled into or near an aquatic environment, environmental weathering processes, such as evaporation and sediment interaction, influence the fate and toxicity of dilbit to aquatic organisms. To date, most studies of the effects of dilbit on the health of aquatic organisms have not considered weathering processes. Thus, the goal of this study was to assess the toxicity of weathered sediment-bound dilbit (WSD) to an aquatic organism. Adult freshwater amphipods (Hyalella azteca) were exposed directly to WSD or the water-soluble fraction (WSF) of WSD. Direct exposure to WSD resulted in oil-mineral aggregates adhering to the appendages and gas exchange structures of amphipods, causing acute lethality. After a 10-min exposure to WSD, amphipods consumed half as much oxygen and their appendage movement was impaired. Exposure to the WSF, which contained a total PAH concentration of 1.08 µg/L, did not result in acute lethality, or significantly affect respiration, activity or acetylcholinesterase activity. Results of the present study indicate that physical interaction with oil-mineral aggregates after a spill of dilbit is a threat to benthic invertebrates, whereas the WSF does not cause acute adverse effects. As the transport of dilbit through pipelines increases in North America, studies must incorporate environmental weathering processes when determining the effects of dilbit on aquatic organisms.