As aquatic invasive species (AIS) proliferate worldwide, a better understanding of their roles in invaded habitats is needed to inform management and introduction prevention strategies and priorities. Metabarcoding of stomach content DNA (scDNA) shows considerable promise in such regard. We thus metabarcoded scDNA from two non-native fish species (alewife (Alosa pseudoharengus) and rainbow smelt (Osmerus mordax)), and three native ones (bloater (Coregonus hoyi), ninespine stickleback (Pungitius pungitius), and slimy sculpin (Cottus cognatus)). Fishes (N = 376) were sampled in spring 2009 and 2010 from 73-128 m depths at three Lake Michigan sites. Four mitochondrial cytochrome oxidase 1 (CO1) primer sets designed to target five potential AIS prey, and a universal aquatic invertebrate CO1 primer set targeting both native and AIS prey were used. Quality controlled prey amplicons were matched to three AIS prey: Bythotrephes longimanus (mean percent frequency occurrence, all samples = 7%), Cercopagis pengoi (5%), and Dreissena rostriformis bugensis (11%). Neither invasive prey Dreissena polymorpha nor Hemimysis anomala were detected. Native prey Leptodiaptomus sicilis, Limnocalanus macrurus, and Mysis diluviana were relatively common in scDNA (respective mean percent occurrences, all samples: 48%, 25%, 42%). Analysis of variation in prey occurrences for sample site, predator species, sample year, sample depth, and predator total length (TL) indicated site and predator species were most important. However, B. longimanus occurrence in scDNA depended upon predator TL, perhaps indicative of its unique defensive spine limiting susceptibility to predation until fishes exceed species-specific gape-based limitations. Our analysis of native and invasive prey species indicated possible indirect AIS impacts such as native predators switching their diet due to AIS-driven losses of preferred native prey. Metabarcoding demonstrated that AIS are integrated components of the offshore Lake Michigan food web, with both native and non-native predators, and both invasive and native prey are affecting species interactions across multiple trophic levels.