Spinosad is a naturally derived insecticide that has shown potential as a mosquito larvicide. To determine the activity of spinosad against blackflies, late-instar larvae from a community comprising Simulium triittatum (63.6%) and seven other species, including three known vectors of onchocerciasis in Mexico (S. metallicum, S. ochraceum, and S. callidum), were subjected to concentration-mortality laboratory bioassays following World Health Organization guidelines. Cephalic capsule measurements confirmed the relatively homogeneous distribution of experimental larvae. The 50% lethal concentration of spinosad was estimated at 1.48 ppm spinosad (95% confidence interval: 1.07–2.33) for a 10-min exposure period, whereas larvae treated with 0.05 ppm of the organophosphate temephos experienced 61% mortality. Immature aquatic insects were identified to genus and tested for their susceptibility to spinosad in the laboratory. After exposure to 12 ppm spinosad for 10 min, ephemeropterans, odonates, trichopterans, and hemipterans did not experience significantly increased mortality over that of untreated controls, whereas a significant increase in mortality was observed in spinosad-treated Plecoptera (P < 0.001). Tilapia and trout fry exposed to 12 ppm spinosad for 10 min did not experience increased mortality at 24-h postexposure over that of the controls. We conclude that spinosad is less toxic than temephos to these blackfly species, but is likely to have a low impact on nontarget members of the aquatic community.