A root extract of Valeriana jatamansi (code BAL-O) exhibited larvicidal and adulticidal activity against different mosquito species. The median lethal concentration (LC₅₀) of BAL-O against larvae of Anopheles stephensi, Anopheles culicifacies, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus were 68.1, 42.8, 51.2, 53.8, and 80.6 mg/liter, respectively. The LC₅₀ and the 90% lethal concentration against adult An. stephensi, An. culicifacies, Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus were 0.14, 0.16, 0.09, 0.08, and 0.17 and 0.24, 0.34, 0.25, 0.21, and 0.28 mg/cm², respectively. The median knock-down time and 90% knock-down time of the fraction were 13, 13, 12, 13, and 18 and 24, 25, 21, 20, and 42 min against An. stephensi, An. culicifacies, Ae. aegypti, An. albopictus and Cx. quinquefasciatus, respectively, using 0.28 mg/cm² impregnated papers. The median effective time and 90% effective time against An. stephensi at 4°C were 46.6 and 8.7 days, and at 29°C, 25.5 and 5.6 days, respectively. Gas chromatographic–mass spectrometric analysis of BAL-O showed 2-butanone,4-(2,6,6-trimethyl-2-cyclohexen-1-yl) (10.11%), patchouli alcohol (8.55%), cubenol (5.97%), caryophyllene oxide (5.46%), cadinol (5.23%), and aristolene (5.19%).