The aim of the present study was to evaluate the activity of pacharin isolated from the ethanol extract from roots of Bauhinia acuruana on third-instar larvae of Aedes aegypti Linn. (Diptera: Culicidae). The crude ethanol extract showed larvicidal activity at the concentration of 500 μg/mL. Given this larvicidal activity, this extract was submitted to chromatographic fractionation on a silica gel column eluted with n-hexane, dichloromethane, ethyl ether, ethyl acetate, and methanol in order to isolate the active compound(s). Pacharin, obtained in pure form from fraction eluted with ethyl ether, was evaluated for their larvicidal effects against A. aegypti. In these bioassays, the larvae were exposed at concentrations of 500, 250, 100, 50, and 25 μg/mL of the crude ethanol extract or pacharin. After 24 h, the number of dead larvae was counted and the LC₅₀ values for larval mortality were calculated. Pacharin showed LC50 value of 78.9 ± 1.8 μg/mL. The structure of isolated compound was identified on the basis of their spectral data (IR, 1D- and 2D-NMR) and by comparison with literature spectral data. The results indicate pacharin as a potential natural larvicide.