Mysids (Mysis diluviana) and dreissenids (Dreissena polymorpha and mostly D. bugensis) are important invertebrate taxa in the food webs of the Laurentian Great Lakes but there are uncertainties about the seasonal and spatial variability in their stable isotope signatures. We quantified δ¹³C and δ¹⁵N in 304 mysid and 366 dreissenid samples across five spatial ecoregions, varying site depth, and three seasons (spring, summer, and fall) in Lake Ontario in 2012 and 2013. Particulate organic matter (POM) was also collected across site depth and season from the Deep Hole ecoregion for use as an isotopic baseline. Lipid normalization models for δ¹³C were generated for both taxa to reduce lipid bias in our statistical analysis. Season was a significant predictor of POM stable isotopes, with δ¹³C lower in the summer and δ¹⁵N decreasing from spring to summer before increasing into fall. Mysid lipid normalized δ¹³C varied by site depth and ecoregion while δ¹⁵N decreased across season and did not vary by site depth or ecoregion. Dreissenid stable isotopes varied significantly across season, depth, and ecoregion, with site depth having positive relationship with δ¹⁵N. Mysids and dreissenids were two trophic positions higher than POM based on δ¹⁵N; this comparison was restricted to the one region where POM was collected. Isotopic variability suggested selective feeding within POM and differing trophic pathways between mysids and dreissenids. Collecting an appropriate taxon across all observed variables to serve as an isotopic baseline, particularly in spatial and temporal studies, is critical to the correct interpretation of trophic relationships.