The metabolic stimulation induced by abiotic stress is an efficient strategy for the production of secondary metabolites in sterile and controlled plant cell, tissue, and organ cultures. Paclitaxel (taxol), one of the most widely used therapeutic compounds for the treatment of various cancers, is mainly produced through cell culture of the European yew (Taxus baccata L.). In this work, a T. baccata callus culture was subjected to drought stress induced in vitro by mannitol and sorbitol (1, 2, 3, and 4%) and sucrose (6 and 8%) to evaluate its impact on physiological and biochemical traits as well as paclitaxel and 10-deacetyl baccatin III (10-DBA) production. It was observed that drought stress caused a significant increase (P < 0.05) in dry weight, proline, soluble sugars, hydrogen peroxide, lipid peroxidation, total phenolics, flavonoids, and flavonols, and a decrease in relative water content, fresh weight, relative growth rate, and cell viability. Constitutive activities of superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase were enhanced by the induced stress. The content of paclitaxel and 10-DBA was higher in stressed cultures than in the control. It can be concluded that T. baccata cells have a protection mechanism against oxidative damage involving induced activities of enzymatic and non-enzymatic antioxidants. The induction of moderate drought stress could therefore be an effective strategy for increasing taxanes production in T. baccata callus cultures.