Chemosynthetic environments support distinct benthic communities capable of utilizing reduced chemical compounds for nutrition. Hundreds of methane seeps have been documented along the U.S. Atlantic margin (USAM), and detailed investigations at a few seeps have revealed distinct environments containing mussels, microbial mats, authigenic carbonates, and soft sediments. The dominant mussel, Bathymodiolus childressi, contains methanotrophic endosymbionts but is also capable of filter feeding, and stable isotope analysis (SIA) of mussel-shell periostracum suggests that these mussels are mixotrophic, assimilating multiple food resources. However, it is unknown whether mixotrophy is widespread or varies spatially and temporally. We used SIA (δ¹³C, δ¹⁵N, and δ³⁴S) and an isotope mixing model (MixSIAR) to estimate resource contribution to B. childressi and characterize food webs at two seep sites (Baltimore Seep; 400 m and Norfolk Seep; 1500 m depths) along the USAM, and applied a linear mixed-effects model to explore the role of mussel population density and tissue type in influencing SIA variance. After controlling for location and temporal variation, isotopic variability was a function of proportion of live mussels present and tissue type. Isotopic differences were also spatially discrete, possibly reflecting variations in the underlying carbon source at the two sites. Low mussel δ¹³C values (∼−63‰) are consistent with a dependence on microbial methane. However, MixSIAR results revealed mixotrophy for mussels at both sites, implying a reliance on a mixture of methane and phytoplankton-derived particulate organic material. The mixing model results also reveal population density-driven patterns, suggesting that resource use is a function of live mussel abundance. Mussel isotopes differed by tissue type, with gill having the lowest δ¹⁵N values relative to muscle and mantle tissues. Based on mass balance equations, up to 79% of the dissolved inorganic carbon (DIC) of the pore fluids within the anaerobic oxidation of the methane zone is derived from methane and available to fuel upper slope deep-sea communities, such as fishes (Dysommina rugosa and Symphurus nebulosus), echinoderms (Odontaster robustus, Echinus wallisi, and Gracilechinus affinis), and shrimp, (Alvinocaris markensis). The presence of these seeps thereby increases the overall trophic and community diversity of the USAM continental slope. Given the presence of hundreds of seeps within the region, primary production at seeps may serve as an important, yet unquantified, energy source to the USAM deep-sea environment.