Mercury contamination in river systems due to historic and current Hg releases is a persistent concern for both wildlife and human health. In larger rivers, like the Ohio River, USA, it is difficult to directly link Hg discharges to bioaccumulation due to the existence of multiple industrial Hg sources as well as the varied dietary and migratory habits of biota. To better understand how industrial effluent influences the cycling and bioaccumulation of Hg within the Ohio River, Hg stable isotope analysis was applied to various nonbiological and biological media. High Hg concentrations in suspended particulate matter suggest this vector was the largest contributor of Hg to the water column, and distinct Hg source signatures were observed in effluent particulates from different industrial processes, such as chlor‐alkali activity (δ²⁰²Hg = −0.52‰) and coal power plant discharge (δ²⁰²Hg = −1.39‰). Despite this distinction, average sediments (δ²⁰²Hg = −1.00 ± 0.23‰) showed intermediate isotopic signatures that suggest the accumulation of a mixed Hg source driven by multiple industrial discharges. Biota in the system were shown to have a conserved range of δ²⁰²Hg and estimation approaches related these signatures back to particulate matter within Hannibal Pool. Mussels were found to conserve Hg isotopes signatures independently of food web drivers and served as ideal water column indicators of bioaccumulated Hg sources. This study highlights the complexity of Hg cycling within an industrialized river and shows that an isotope tracer approach can provide insight to water column sources of Hg. Integr Environ Assess Manag 2021;17:233−242. Published 2020. This article is a US Government work and is in the public domain in the USA.