Grapevine trunk diseases (GTDs) are taking the forefront of winegrowers’ concerns, as they cause considerable damage in vineyards not only quantitatively but also qualitatively. Furthermore, in the last three decades, an increasing incidence of the socioeconomic impact of these fungal pathologies has been observed. To date, no effective control strategies or curative treatments are available for these diseases that can replace the effectiveness offered by a series of chemical synthesis fungicides currently prohibited by European legislation. In this scenario, screening for less sensitive cultivars is regarded as a sustainable approach for GTDs management. In the study presented herein, the tolerance/susceptibility of 25 cultivars from the Grapevine Germplasm Bank of Aragon (Movera, Zaragoza, Spain) including commercial, local, or minority germplasm, was tested against two pathogens associated with Botryosphaeria dieback (viz. Neofusicoccum parvum and Diplodia seriata), which were inoculated in a detached cutting assay under open-air conditions. Based on lesion length development after eight months, significant differences were detected among the cultivars in the length of internal (vascular) necroses. In general terms, all cultivars were susceptible to fungal infection, but ‘Macabeo’ and one of the ‘Garnacha Tinta’ ecotypes under study (from Villanueva de Huerva, Zaragoza) would be the least susceptible white and red cultivars, respectively. On the other hand, ‘Monegrina’, ‘Grumel’, and ‘Torcijón’ would be among the least tolerant cultivars to fungal infection.