Acclimation of plants to water deficit involves biochemical and physiological adjustments. Here, we studied how UV-B exposure and exogenously applied hydrogen peroxide (H2O2) potentiates drought tolerance in tobacco (Nicotiana tabacum L.). Separate and combined applications for 14 days of 1.75 kJ m−2 day−1 UV-B radiation and 0.2 mM H2O2 were assessed. Both factors, individually and combined, resulted in inhibition of growth. Furthermore, the combined treatment led to the most compacted plants. UV-B- and UV-B+H2O2-treated plants increased total antioxidant capacity and foliar epidermal flavonol content. H2O22- and UV-B+H2O2-pre-treated plants showed cross-tolerance to a subsequent 7-day drought treatment. Plant responses to the pre-treatment were notably different: i) H2O2 increased the activity of catalase, phenylalanine ammonia lyase and peroxidase activities, and ii) the combined treatment induced epidermal flavonols which were key to drought tolerance. We report synergistic effects of UV-B and H2O2 on transcription accumulation of UV RESISTANCE LOCUS 8, NAC DOMAIN PROTEIN 13 (NAC13), and BRI1-EMS-SUPPRESSOR 1 (BES1). Our data demonstrate a pre-treatment-dependent response to drought for NAC13, BES1 and CHALCONE SYNTHASE transcript accumulation. This study highlights the potential of combining UV-B and H2O2 to improve drought tolerance which could become a useful tool to reduce water use.

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