The general aim of this work was to compare the leaf-level responses of different protective components to water deficit and high temperatures in Quercus cerris L. and Quercus robur L. Several biochemical components of the osmotic adjustment and antioxidant system were investigated together with changes in hormones. Q. cerris and Q. robur seedlings responded to water deficit and high temperatures by: (1) activating a different pattern of osmoregulation and antioxidant mechanisms depending on the species and on the nature of the stress; (2) upregulating the synthesis of a newly-explored osmoprotectant, dimethylsulphoniopropionate (DMSP); (3) trading-off between metabolites; and (4) modulating hormone levels. Under water deficit, Q. cerris had a higher antioxidant capacity compared to Q. robur, which showed a lower investment in the antioxidant system. In both species, exposure to high temperatures induced a strong osmoregulation capacity that appeared largely conferred by DMSP in Q. cerris and by glycine betaine in Q. robur. Collectively, the more stress-responsive compounds in each species were those present at a significant basal level in non-stress conditions. Our results were discussed in terms of pre-adaptation and stress-induced metabolic patterns as related to species-specific stress tolerance features.