The Little St. Francis River and its tributaries drain metals-contaminated areas of the Madison County Mines National Priority List Superfund site (MCM) which was designated in 2003 to facilitate remediation of metals contamination within the MCM. One concern for natural resource trustees in the MCM is the potential effects of elevated metals concentrations on the federally threatened St. Francis River crayfish, Faxonius quadruncus, which has a geographic range that is limited to the St. Francis River watershed. A survey of riffle-dwelling crayfish, in-situ cage study, and laboratory toxicity tests were conducted to assess the effects of mining-derived metals on F. quadruncus and other crayfish species in the MCM. Crayfish densities were significantly greater at sites upstream of metals releases from historical mining (henceforth mining releases) compared to densities at sites downstream of mining releases, and metals concentrations in whole-body crayfish, surface water, sediments, macroinvertebrates, fish, and plant material were greater at sites downstream of mining releases compared to sites upstream of mining releases. Crayfish densities were also negatively correlated with consensus-based adverse effects indices, expressed as surface-water toxic units and sediment probable effects quotients. Decreased growth and increased mortality during cage and laboratory studies were likely due to exposure to, and subsequently uptake of, elevated concentrations of metals. Crayfish in all studies were found to bioaccumulate metals, which supports their utility as bioindicators of metals contamination. Study results show that elevated metals concentrations associated with mining releases in the MCM continue to adversely affect biota, including the federally threatened F. quadruncus.