Former nuclear weapons material production at the U.S. Department of Energy's Savannah River Site (SRS) has resulted in contamination of certain terrestrial and aquatic ecosystems on site with legacy wastes such as radiocesium (137Cs), tritium (3H), and metals. We collected fish and invertebrates from five beaver ponds (sites) above, adjacent, and downgradient of three SRS facilities (H-, F-, and C-Areas) to evaluate whether the accumulation of metals and radionuclides in biota were associated with specific facility operations and if the measured levels could pose risks to aquatic organisms. We compared concentrations of various metals, 137Cs, and 3H in fish, as well as in water (3H only), among sites along the stream gradient. Fish collected from sites adjacent to H-Area had significantly higher 137Cs concentrations compared to fish from other sites. Both biota and water samples indicated significantly greater levels of 3H in sites adjacent to and downstream of C-Area. Concentrations of zinc (Zn), copper (Cu), and mercury (Hg) in some samples exceeded effects levels reported for fish and may pose a risk to fish populations. This study reported fish tissue concentrations of 137Cs and 3H, which have not been documented extensively in ecotoxicological studies. Our results suggested that industrial operations such as nuclear material production at SRS could have long-lasting impact on the aquatic ecosystem via the release of radionuclides and metals, and long-term monitoring of physiological effects and population level impact in biota exposed to these contaminants are recommended.