Updated project metadata. Neuropteran larvae are fierce predators that use venom to feed on arthropods, but their venom system is poorly understood. The iconic antlions (Myrmeleontidae) are adapted to dry, sandy habitats, where they use pitfall traps and venom to catch and subdue large arthropod prey. The aphid lions, larvae of green lacewings (Chrysopidae) primarily hunt for smaller prey, such as aphids. Here, we investigate the tissue origin, composition and ecological role of venom in the European antlion Euroleon nostras and the green lacewing Chrysoperla carnea. We show that E. nostras venom has potent insecticidal activity, indicating its importance for efficient immobilization of large prey. C. carnea venom exerted no toxic effects, reflecting their specialization to small, non-defensive prey. Histological and proteotranscriptomic analysis revealed that antlion venom is produced in three distinct glands that secrete different proteins, indicating a functional compartmentalization of venom compounds. The venom system of E. nostras contains no bacteria, providing strong evidence that all venom compounds are of insect origin and not produced by bacterial symbionts as previously suggested. Our study provides a detailed analysis of the neuropteran venom system and new insights into venom biochemistry and function that may underpin the adaptation of antlions to their unique ecological niche.