The trophic niche of invasive species can vary overtime because of different processes related to ecological opportunity and invader activity that condition biological interactions with the native biodiversity. We conducted an annual-based survey of the trophic niche of the mysid Hemimysis anomala in the largest European peri-alpine lake by combining molecular and isotope analyses. We hypothesized that the population trophic niche width would vary seasonally, expanding in warm periods due to greater ecological opportunities and higher mysid metabolic activity. Molecular analyses identified a diversified set of prey throughout the year ranging from autotrophic protists to zooplankton and converged with isotope analyses to support the diet seasonality hypothesis of H. anomala with wider trophic niches and, in a lower extent, richer diets in spring and summer when compared to autumn and winter. Isotope analyses also highlighted a seasonal pattern in resources used with a dominance of pelagic reliance in summer and autumn. These results underlined the significant seasonal variability in the trophic niche of this invasive mysid suggesting that future assessments of its ecological impacts should account for the temporal variations of its trophic interactions with the native biodiversity.