Traditional chemical pesticides pose potential threats to human health, the environment, and food safety, and there is an urgent need to develop botanical pesticides that are easily degradable, renewable, and environmentally compatible. This research serves to detect the lethal impacts of Amanita pantherina(DC.:Fr) Schrmm.(Agaricales, Amanitaceae, Amanita), Amanita virgineoides Bas (Agaricales, Amanitaceae, Amanita), Coprinus comatus (O.F.Müll.) Pers. (Agaricales, Psathyrellaceae, Coprinus), Pycnoporus cinnabarinus(Jacq.:Fr) Karst (Polyporales, Polyporaceae, Polyporus) and Phallus rubicundus (Bosc) Fr. (Phallales, Phallaceae, Phallus) on Drosophila melanogaster(Diptera, Drosophilidae, Drosophila), including their effects on lifespan, fecundity, offspring growth and developmental characteristics, antioxidant enzyme activity, peroxide content, and the gene transcription associated with signaling pathways and lifespan of D. melanogaster. The results demonstrated that they all produced lethal effects on D. melanogaster. Female flies were more sensitive to the addition of macrofungi to their diet and have a shorter survival time than male flies. The toxic activity of A. pantherina-supplemented diet was the strongest, so that the D. melanogaster in this group had no offspring. The macrofungal-supplemented diets were able to significantly reduce the activity of antioxidant enzymes, accumulate peroxidation products, up-regulatd the transcription of genes related to signaling pathways, inhibit the expression of longevity genes, reduce the lifespan and fertility of D. melanogaster. Consequently, we hypothetically suggest that medicinal C. comatus, P. cinnabarinus and P. rubicundus hold the potential to be developed into an environmentally friendly biopesticide for fly killing.