The gall midge Lasioptera donacis, whose larval stage interferes with the reed’s leaf development, is a potential candidate agent for the biological control of Arundo donax. Reed infestation is always associated with the presence of a saprophytic fungus, Arthrinium arundinis, which is believed to provide food for the larvae. Larvae also interact with a parasitic nematode, Tripius gyraloura, which can be considered its natural enemy. To deepen our knowledge of the plant–fungus–insect trophic interactions and to understand the effects of the nematode on midge larval feeding behaviour, we applied stable isotope analysis, one of the most effective methods for investigating animal feeding preferences in various contexts. The results showed that on average the fungus accounted for 65% of the diet of the midge larvae, which however consumed the reed and the fungus in variable proportions depending on reed quality (expressed as the C:N ratio). No differences in feeding behaviour were observed between parasitised and non-parasitised midge larvae, indicating that nematodes have no effect in this regard. Due to its trophic habits, L. donacis could be an effective control agent of A. donax and these results need to be considered when implementing biological control measures.