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ABSTRACT Freshwater ecosystems are globally threatened by multiple climatic and anthropogenic stressors, including facilitated biological invasion and eutrophication, and thus understanding their effects on ecosystem function is critical. One key aspect is the availability of long‐chain polyunsaturated fatty acids (LC‐PUFAs), which are essential for the development and reproduction of aquatic organisms. Biological invasion can reshape trophic links and restructure food web energy pathways, while eutrophication can affect community composition as well as LC‐PUFAs availability, especially in food webs. Using a space‐for‐time approach, we investigated how these stressors influenced food web energy pathways and nutritional quality in fluvial lakes of the Upper St. Lawrence River (Canada). We aimed to (1) investigate how invasive dreissenid mussels ( Dreissena polymorpha and Dreissena bugensis ) and invasive round goby ( Neogobius melanostomus ), modify energy pathways and nutritional quality, and whether these changes cascade to native piscivorous fish; (2) assess whether these invaders alter nutriment enrichment effects on LC‐PUFAs content in the food web. We sampled food web components, including seston, macroinvertebrates, and fish from one uninvaded and two invaded fluvial lakes. We employed stable isotope analyses supported by stomach content analyses to describe food web energy pathways. Fatty acids were applied as an indicator of food web quality, while both fatty acids and relative weight indices were used to assess fish body condition. In invaded lakes, smallmouth bass ( Micropterus dolomieu ), and large yellow perch ( Perca flavescens ) (> 100 mm) had a lower proportion of pelagic items in their diet than those in the uninvaded lake. Smallmouth bass, which fed on invasive round gobies, exhibited high relative weights in the invaded lake but also showed altered fatty acid compositions and a reduced proportion of LC‐PUFAs. No patterns were observed for large yellow perch. Phosphorus concentrations were negatively associated with LC‐PUFAs content in seston and round goby but showed no relationship in most macroinvertebrates and benthivorous small yellow perch (< 100 mm). Our study highlights how invasive species disrupt food web energy dynamics by altering pelagic‐benthic pathways. In particular, the interplay between invasive species and nutriment enrichment can influence LC‐PUFAs and reduce food web quality.