Objective: To evaluate the acaricidal activity of the essential oil obtained from roots of Derris floribunda (D. floribunda) (Miq.) Benth, and its main constituent nerolidol against the Mexican mite Tetranychus mexicanus (T. mexicanus) (McGregor). Methods: The essential oil from the roots of D. floribunda collected in the Amazon region (Brazil) was obtained by hydrodistillation. Its chemical composition was determined by GC–MS analysis. The acaricidal activities of this essential oil and nerolidol, were evaluated by recording the number of dead females (mortality) and eggs (fertility). Results: The essential oil showed sesquiterpenes as major volatile components. Nerolidol, the main component, represented 68.5% of the total composition of the essential oil. D. floribunda essential oil and nerolidol showed acaricidal activity, with LC₅₀ of 9.61 μg/mL air and 9.2 μg/mL air, respectively, over a 72 h period. In addition, both the essential oil and nerolidol significantly reduced the fecundity of T. mexicanus. Conclusions: Due to the economic importance of T. mexicanus and the lack of new pesticides, our data are very promising in the search for efficient and safer acaricidal products. Furthermore, this is the first report about the chemical composition and bioactivity of the essential oil of the Amazon plant species D. floribunda.