As urbanization rapidly expands, the divide between anthropogenic activities and naturally occurring wildlife species is becoming increasingly blurred. Many mesopredators, including the North American river otter (Lontra canadensis), exhibit behavioral flexibility in response to the unique selective pressures in urban landscapes. Despite the occurrence and apparent success of river otters in urban areas in the United States, to our knowledge, there are no published studies of the impacts of urbanization on the plasticity of the species’ diet. We collected 105 river otter spraints from six stream reaches across Mecklenburg County, North Carolina, USA to investigate diet composition using morphological analysis and eDNA metabarcoding. We used generalized linear mixed-effects models (GLMM) and redundancy analyses (RDA) to test for the effects of landscape urbanization, stream discharge, water quality, and seasonality on prey species richness and community composition. None of the predictor variables had significant effects on prey species richness. However, all predictor variables significantly influenced prey community composition. Diet characterization from morphological and molecular analysis of spraints revealed that river otters primarily consumed fish (Perciformes, Siluriformes, Cyprinodontiformes), crayfish (Procambarus, Faxonius, Cambarus), and amphibians (Lithobates), as well as with mammals, reptiles, birds, and non-crayfish macroinvertebrates. These results support the hypothesis that river otters are true opportunists, taking advantage of readily available prey across different environmental contexts. We recommend that future research explore additional water quality parameters, assess how urbanization influences the nutritional quality of available prey, and investigate patterns of otter movements in urban landscapes.