The fast and remarkable growth of global aquaculture in recent years has created new challenges, such as guaranteeing a sustainable supply of raw materials used for aquafeed formulation. Gammarids are low-trophic crustaceans with an increasing interest in aquaculture due to their high nutritional profiles and their capacity to grow under high-density conditions. Moreover, gammarids have the ability to thrive on a wide range of sidestreams while accumulating relatively high levels of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA). In the present study, juveniles of the marine gammarid Gammarus locusta were cultured at four different temperatures (5°C, 10°C, 15°C, and 20°C) for 21 days and fed three diets, including the seaweed Fucus sp. as control, and carrot leaves and coconut flesh representing two agri-food industry sidestreams. Our results indicate that both the survival and biomass of G. locusta were highly affected by diet, with coconut showing the lowest growth performance. The temperature had no effect on biomass, although high temperature (20°C) resulted in a decrease in survival. The effects of temperature on the gammarid fatty acids were not evident, with diet being the main modulator of the profiles. Furthermore, the results also reveal that the Fucus sp. diet was associated with relatively high percentages of n-3 and n-6 LC-PUFA. Interestingly, essential LC-PUFA such as eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic (22:6n-3, DHA) acids were detected in gammarids fed on either Fucus sp. or any of the sidestreams irrespectively of their presence in the diets. These results suggest an ability of G. locusta for LC-PUFA biosynthesis (trophic upgrading) and/or retention, making this species a promising candidate for the production of high-value ingredients for aquafeeds.