Aims: To evaluate the capacity of Oenococcus oeni strains to release aroma compounds from glycosylated precursors by measuring glycosidase activities with both synthetic and natural substrates. Methods and Results: Five glycosidase activities were investigated in 47 O. oeni strains using synthetic substrates. This screening revealed that activity levels vary considerably, not only for each strain (depending on the substrate tested), but also between strains. Fifteen strains exhibiting different activity profiles were further analysed using natural substrates extracted from both untoasted and toasted oak. In the latter, various amounts of aromatic compounds were measured, thus confirming the specific potentials of the selected strains, but the results were different from those obtained using synthetic substrates. In addition, the use of toasted wood extracts significantly increased the release of wood aromas, which minimized differences between strains. Conclusions: The capability of O. oeni to hydrolysate glycoconjugate aroma precursors is strain-dependent and variable, depending on the substrate. Significance and Impact of the Study: Instead of synthetic substrates, natural aroma precursors should be used for an adequate evaluation of the glycosidase potential of O. oeni.