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Long-lasting insecticidal nets and indoor residual sprays have played a major role in significantly reducing the burden of malaria. However, the management of mosquitoes resistant to current insecticides continues to be challenging. A promising new strategy is drug-based vector control where drugs are administered to vector hosts (human/animal), which renders the host blood toxic to mosquitoes—thereby reducing mosquito lifespan, fecundity and onward transmission of pathogens. Afoxolaner, fluralaner, and lotilaner are isoxazoline derivatives widely used as systemic insecticides for ectoparasite control in companion animals. Here, the mosquitocidal activity of these compounds against Anopheles gambiae s.s, Anopheles coluzzii , Aedes aegypti , and Anopheles funestus mosquitoes was evaluated. The effect of antibiotic treatment and different carbohydrates in artificial nectar meals on isoxazoline activity was also investigated. All isoxazolines tested showed rapid killing with little difference in susceptibility between different species and genera of mosquitoes. Fluralaner showed the most potent mosquitocidal effects with IC 50 values ranging from 44 to 87 nM when mortality was assessed at 24 h post-feeding. Addition of the antibiotic cocktail or variations in sugar diet did not affect isoxazoline potency. In conclusion, data indicate potent and rapid mosquitocidal effects of isoxazolines that are likely unaffected by mosquito sugar feeding behaviour and microbiome dynamics.