Re-emergence of Grapevine Trunk Diseases (GTDs) in many world vineyards within the span of a mere two decades has become a subject of major concern for the viticulture sector. In Tunisia, knowledge about the symptoms of GTDs and the associated microflora is hitherto incomplete. In order to investigate this point, ten vineyards served as model for the analysis and monitoring of the symptoms of GTDs on cv. Italia (Muscat d’Italie) used to produce table grapes, in the Northern region of Tunisia. Depending on the vineyards, typical GTD-foliar symptoms with incidence ranging from 0.03% to 6% were observed. Fungal strains were isolated from necrotic wood tissues of the trunk of grapevines showing decline, small and distorted leaves and chloroses. Sequencing of the rDNA-Internal Transcribed Spacer region of the fungal strains allowed us to identify for the first time in Tunisia three fungi described in the literature as involved in GTDs: Lasidiodiplodia pseudotheobromae, Neofusicoccum parvum and Schizophyllum commune. The ability of these fungal strains to grow in Mediterranean climate was investigated by studying mycelial growth at various temperatures. As these fungal strains were isolated from the same wood samples, their ability to compete in vitro and in planta was tested, as well as their pathogenicity. For that purpose, strains were inoculated singly or in combination on cv. Italia young vines. Pathogenicity of the isolated fungi was evident but positive interaction between fungal strains was not observed.