Grapevine trunk diseases (GTDs) have become a major concern to viticulture worldwide, as they have a significant economic impact on the longevity and productivity of vineyards. The simultaneous presence of several trunk pathogens in a single plant with inconsistent expression of symptoms, together with their isolation in asymptomatic grapevines and the lack of effective treatments, make these diseases extremely complex to identify and eradicate. In order to improve the knowledge on GTDs and to search for new sustainable alternatives to limit their development, the structure of fungal communities associated with GTD-infected symptomatic and asymptomatic plants was studied. Firstly, 10cm long spurs were collected from two selected grapevine cultivars with different levels of susceptibility to GTDs, in three different locations belonging to the Alentejo region (southern Portugal), and this was followed by a metagenomic analysis. Deep sequencing of fungal-directed ITS1 and ITS2 amplicons led to the detection of 1923 ASVs in the grapevine fungal microbiota, where 393 different fungal genera were found, including 29 fungal genera/species previously described as responsible for GTDs. Our results revealed that the structure of the fungal communities of the two cultivars from the three sampling sites followed a similar trend in terms of plant symptomatology. However, there were statistically significant differences in fungal abundance according to vineyard location. Regarding trunk pathogens, there are significant differences associated with the expression of trunk diseases symptoms, cultivar, and vineyard location. Our study allowed an adequate sequencing depth to unravel the complexity of the grapevine fungal communities of the selected cultivars and updated the information on the richness and diversity of GTDs-associated fungi and their relationship with the plant symptomatology. This study contributes to a better understanding of plant–pathogen interactions and to a better knowledge of GTDs, helping to mitigate and control these diseases with such a high economic impact worldwide.