The screening of Chinese medicinal herbs for insecticidal principles showed that the essential oil of Echinops grijsii Hance roots possessed significant larvicidal activity against mosquitoes. The essential oil was extracted via hydrodistillation and its constituents were determined by gas chromatography‐mass spectrometry (GC‐MS) analysis. GC‐MS analyses revealed the presence of 31 components, with 5‐(3‐buten‐1‐yn‐1‐yl)‐2,2′‐bithiophene (5‐BBT, 27.63%), αterthienyl (α‐T, 14.95%),1,8‐cineole (5.56%) and cis‐β‐ocimene (5.01%) being the four major constituents. Based bioactivity‐directed chromatographic separation of the essential oil led to the isolation of 5‐BBT, 5‐(4‐isovaleroyloxybut‐1‐ynyl)‐2,2′‐bithiophene (5‐IBT) and αT as active compounds. The essential oil of E. grijsii exhibited larvicidal activity against the fourth instar larvae of Aedes albopictus, Anopheles sinensis and Culex pipiens pallens with LC50 values of 2.65 μg/mL, 3.43 μg/mL and 1.47 μg/mL, respectively. The isolated thiophenes, 5‐BBT and 5‐IBT, possessed strong larvicidal activity against the fourth instar larvae of Ae. albopictus(LC50 = 0.34 μg/mL and 0.45 μg/mL, respectively) and An. sinensis(LC50 = 1.36 μg/mL and 5.36 μg/mL, respectively). The two isolated thiophenes also had LC50 values against the fourth instar larvae of C. pipiens pallens of 0.12 μg/mL and 0.33 μg/mL, respectively. The findings indicated that the essential oil of E. grijsii roots and the isolated thiophenes have an excellent potential for use in the control of Ae.albopictus, An. sinensis and C. pipiens pallens larvae and could be used in the search for new, safer and more effective natural compounds as larvicides.