Every year Chile exports about 2000 tons of boldo folium ( Peumus boldus ), which is used around the world as a traditional herbal medicinal product (THMP), mostly to relieve gastrointestinal disorders. This biomass may be a resource for the agrochemical industry to manufacture botanical insecticides. In this regard, the insecticidal potential of boldo has been poorly investigated. In the present work, hydrodistillation of a commercial boldo folium gave 1.5% ( w / w ) of a yellowish essential oil (boldo essential oil, BEO) containing 1,8-cineole (20.7%), p -cymene (18.5%), limonene (9.1%), ascaridole (9.1%) and β-phellandrene (6.4%) as the main constituents, as determined by gas chromatography-mass spectrometry (GC-MS). NMR analysis allowed us to determine that ascaridole was mainly represented by the cis -isomer. BEO was toxic to larvae of the filariasis vector Culex quinquefasciatus and adults of the housefly Musca domestica , showing LC50/LD50 values of 67.9 mg · L−1 and 98.5 µg · adult−1, respectively. On the other hand, lower insecticidal activity was observed against larvae of the moth pest Spodoptera littoralis (LD50 of 268.9 µg · larva−1). It is worth noting that, when tested at LC90 concentration, BEO was significantly less toxic to aquatic microcrustacean Daphnia magna than the conventional insecticide α-cypermethrin. Finally, in the attempt to explore the BEO mode of action, we tested it for acetylcholinesterase (AChE) inhibitory properties using the Ellman method, obtaining negligible effects (IC50 = 0.45 mg·mL−1). Taken together, these results gave new insights into the potential of BEO as a future ingredient of botanical insecticides.