Gut content analysis (GCA), stable isotopes analysis (SIA), and the multiple sources MixSIR mixing model were used to evaluate the dietary niches of 5 important species in Lake Annecy fisheries. Observations on gut content data revealed a variety of feeding habits, with some potential diet overlap between European whitefish (Coregonous lavaretus), Arctic char (Salvelinus alpinus), and Eurasian perch (Perca fluviatilis) during the winter and spring. However, food resources seemed to be partitioned among the Lake Annecy fish community during summer growth period, possibly limiting competitive interactions. Ten years of SIA data were used to evaluate food web dynamics. Values of δ15N indicated very low interspecies variability, whereas δ13C revealed the differences between fish species; pelagic species were δ13C depleted, while littoral species were δ13C enriched. The results demonstrated that Arctic char have a separate sustainable dietary niche and lower predation mortality. Stocking Arctic char to enhance populations might not have an impact on other fish populations. We found that the natural dietary variability can be misinterpreted when using GCA only. Our findings highlight the importance of combining both GCA and SIA to explore the trophic relationships between predators and prey, especially in Lake Annecy having spatial and seasonal variations in prey isotopic signatures. Results from these studies on food web dynamics could have implications in fisheries management decisions and development of trophic models.