With improved analytical techniques, environmental monitoring studies are increasingly able to report the occurrence of tens or hundreds of chemicals per site, making it difficult to identify the most relevant chemicals from a biological standpoint. For this study, organic chemical occurrence was examined, individually and as mixtures, in the context of potential biological effects. Sediment was collected at 71 Great Lakes tributary sites and analyzed for 87 chemicals. Multiple risk-based lines of evidence were used to prioritize chemicals and locations, including comparing sediment concentrations and estimated porewater concentrations to established whole-organism benchmarks (i.e., sediment and water quality criteria and screening values) and to high-throughput toxicity screening data from the U.S. Environmental Protection Agency's ToxCast database, estimating additive effects of chemical mixtures on common ToxCast endpoints, and estimating toxic equivalencies for mixtures of alkylphenols and polycyclic aromatic hydrocarbons (PAHs). This multiple-lines-of-evidence approach enabled the screening of more chemicals, mitigated the uncertainties of individual approaches, and strengthened common conclusions. Collectively, at least one benchmark/screening value was exceeded for 54 of the 87 chemicals, with exceedances observed at all 71 of the monitoring sites. Chemicals with the greatest potential for biological effects, both individually and as mixture components, were bisphenol A, 4-nonylphenol, indole, carbazole, and several polycyclic aromatic hydrocarbons (PAHs). Potential adverse outcomes based on ToxCast gene targets and putative adverse outcome pathways relevant to individual chemicals and chemical mixtures included tumors, skewed sex ratios, reproductive dysfunction, hepatic steatosis, and early mortality, among others. Results provide a screening level prioritization of chemicals with the greatest potential for adverse biological effects and an indication of sites where they are most likely to occur.