Sediments in Lyons Creek East (Welland, Ontario), a tributary of the Niagara River and part of the Niagara River Area of Concern, which exceed screening-level environmental-quality criteria for multiple contaminants, were assessed for biological impacts using information from multiple lines of evidence. An initial chemical survey indicated the primary contaminants of concern to be polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), zinc, and p,p'-DDE due to frequent exceedences of sediment guidelines. A subsequent study focused on the chemical composition of sediment, status of benthic invertebrate communities, contaminant bioaccumulation in resident benthos, and sediment toxicity to laboratory-exposed organisms. Chemical and biological conditions in the creek were compared with those in reference creeks using both multivariate (cluster analysis and ordination) and univariate (regression) techniques. Sediment PCBs (≤ 19 μg/g), PAHs (≤ 63 μg/g), and Zn (≤ 7969 μg/g) were increased above the sediment-quality guidelines along most of the creek; however, the upper 1.5 km portion of the creek was the most highly contaminated and therefore the main focus for biological study. Although severe toxicity was evident at several locations in the upper creek, resident benthic communities were minimally affected by sediment contamination. The cause of toxicity was likely related to a combination of stressors, including PCBs, PAHs, and metals. Due to its biomagnifiable nature, bioaccumulation focused on PCBs; concentrations in resident macroinvertebrates were ≤ 2 orders of magnitude greater than those found in reference creeks and were above tissue residue guidelines, indicating a potential risk for consumers of benthos. This risk was not limited to the upper 1.5 km where other effects were seen.