Mode of action of farnesene, a volatile sesquiterpene commonly found in the essential oils of several plants, was deeply studied on the model species Arabidopsis thaliana. The effects of farnesene on the Arabidopsis root morphology were evaluated by different microscopic techniques. As well, microtubules immunolabeling, phytohormone measurements and ROS staining helped us to elucidate the single or multi-modes of action of this sesquiterpene on plant metabolism. Farnesene-treated roots showed a strong growth inhibition and marked modifications on morphology, important tissue alterations, cellular damages and anisotropic growth. Left-handed growth of farnesene-treated roots, reverted by taxol (a known microtubule stabilizer), was related to microtubule condensation and disorganization. As well, the inhibition of primary root growth, lateral root number, lateral root length, and both root hairs length and density could be explained by the strong increment in ethylene production and auxin content detected in farnesene-treated seedlings. Microtubule alteration and hormonal unbalance appear as important components in the mode of action of farnesene and confirm the strong phytotoxic potential of this sesquiterpene.