Global food supply strongly depends on honeybee pollination services, which are threatened byinsecticides and pests such as parasitic Varroa destructor mites. Chemical varroacides/acaricides arehampered by resistance development, necessitating the development of sustainable andenvironmentally friendly alternatives, with arthropod venom peptides being considered promisingsources of acaricidal toxins. With only a few acaricidal venom peptides being reported, we performed asystematic topical screening of 50 arthropod venoms against V. destructor, with 78% of the venomscausing 100% mortality after 24 h. Deconvolution of the venoms from the Tasmanian cave spiderHickmania troglodytes and the Giant Japanese funnel-web spider Gigathele gigas led to identificationof the varroacidal peptides Ht1a and Gg1a. Topical application of Ht1a and Gg1a reduced varroa mitebut not honeybee survival, despite Ht1a inhibiting voltage-gated sodium channels from varroa andhoneybee with equal potency. Ht1a and Gg1a were inactive against human skeletal muscle (hNa V 1.4),cardiac (Na V 1.5), neuronal Na V channel isoforms, and human voltage-gated calcium channel Ca V 2.2.At human α3β2/4 nicotinic acetylcholine receptors, Gg1a was inactive while 10 μM of Ht1a partiallyblocked nicotine-mediated Ca 2+ influx. Our data reveal Ht1a and Gg1a as promising candidates for thedevelopment of novel varroa mite treatments of honeybee hives.