Australian funnel-web spiders (FWS) are among the most dangerous spiders in the world. The distribution of the 40 described species is restricted to the eastern coast of Australia, and coincides with that of high human population density, leading to frequent and potentially deadly encounters. Most FWS species are ground-dwelling, including the infamous Atrax robustus (or Sydney FWS) or Hadronyche infensa (Toowoomba FWS), whereas the tree-dwelling species are so far limited to only two species, Hadronyche cerberea (southern tree-dwelling FWS) and Hadronyche formidabilis (northern tree-dwelling FWS). All recorded human fatalities have resulted from the bites of the Sydney FWS, but other species can also lead to severe envenomations. In fact, a much higher rate of severe envenomations was reported for tree-dwelling species (~75 %) compared to ground-dwelling species (~15-20 %). Here, we investigate if adaptation to a novel ecological niche (tree) and access to different preys and exposure to novel predators, may have led to the evolution of more potent venom. Through an integrated approach, including venom gland transcriptomics and proteomics coupled to bioassays, the venom composition and biological activity toward insects and mammals was uncovered, providing evidence for increased potency and specific adaptations.