Botryosphaeria dieback is a grapevine trunk disease that affects all viticulture regions of the world. Species of the genus have been reported as pathogenic toward grapevine in several growing regions and have also been previously reported from Portuguese vineyards. Species in this genus, particularly , have been reported in previous studies to be more aggressive than other Botryosphaeriaceae species most commonly associated with Botryosphaeria dieback. The aim of this study was to assess the response of some of the more representative cultivars planted throughout Portuguese vineyards, 'Touriga Nacional,' 'Touriga Franca,' 'Alvarinho,' 'Aragonez' (= 'Tempranillo'), and 'Cabernet Sauvignon,' by performing artificial inoculations with spp. collected in different geographic locations worldwide. Two experiments, one that involved inoculating 2-year-old grapevines kept in greenhouse-controlled conditions with six isolates of and one isolate of and one that involved inoculating 7-year-old field-grown grapevines with two isolates of , were conducted twice. We assessed the response of the cultivars by evaluating the length of lesions caused by the isolates 5 months after inoculation. The results showed that all isolates studied were able to infect the annual shoots because they were always reisolated and produced internal wood discoloration. Significant differences were found for all isolate-cultivar combinations. In both experiments, Touriga Nacional showed the largest lesions and while Aragonez recorded the smallest lesions of the cultivars inoculated with spp. In general, Portuguese isolates were more aggressive than those from Peru, which were mildly aggressive. These results are a first insight into the response of selected Portuguese cultivars to species, which are present in Portugal but not commonly associated with Botryosphaeria dieback. This research contributes to our knowledge of the impact that Botryosphaeria dieback causal agents have on crucial national cultivars, which may help winegrowers not only manage current cultural practices but also optimize decision making when planning new vineyards.