The Ok Tedi mine discharges waste rock and tailings into the Ok Tedi River in Papua New Guinea. This has resulted in elevated copper concentrations throughout the Ok Tedi/Fly River system, which can potentially impact aquatic biota. Ten years of measured copper and toxicity monitoring data were used to assess the risk of chronic effects from the mine-derived copper. Cumulative probability plots of dissolved and labile copper were compared to a species sensitivity distribution (SSD) of published copper toxicity data for four regions of the river. The Cu-SSD was used to estimate the risk of chronic effects to aquatic organisms in the Ok Tedi/Fly River at a range of potential copper exposure scenarios. The risk to species at the median labile copper concentration for each region showed a gradient effect with distance downstream from the mine and only the most sensitive (0.2–11%) species were at risk. There were copper exceedances of the region-specific guideline values (GV) and default guideline value (DGV) 88% and 74% of the time, respectively, in the Ok Tedi region (closest to the mine) and this is considered a high risk of chronic effects. Measured copper concentrations in the middle Fly River, lower Fly River (farthest downstream of the mine) and the river at Kiunga (reference site) exceeded the region-specific GVs and DGVs less frequently to rarely and presented a lower risk of chronic effects from copper. The risk was supported using toxicity tests with the local microalgal species Chlorella sp. Comparison of recent (2010−2020) and historical (1996–2004) copper monitoring data from the Ok Tedi/Fly River indicated a decrease in the labile copper concentrations (30–76%) at key sites from impacted regions and a subsequent decrease in risk. This coincides with improved mining practices aimed at reducing the copper load into the Ok Tedi/Fly River.