Filamentous fungi are the main pathogens of withered grapes destined for passito wine production. Knowledge of which species inhabit these post-harvest fruits and their pathogenicity is essential in order to develop strategies to control infection, but is still scarce. This study investigated the predominant mycobiota of withered grapes through a cultivation-dependent approach. Strain and species heterogeneity was evidenced on examining isolates collected over three consecutive years. Colony morphology and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis revealed the occurrence of several phenotypes and haplotypes, respectively. Strains were phylogenetically analyzed based on sequence typing of different genes or regions (e.g. calmodulin, β-tubulin and internal transcribed spacer region). Beside the most common necrotrophic-saprophytic species of Penicillium, Aspergillus, Alternaria and Botrytis species responsible for fruit rot, other saprobic species were identified (e.g. Trichoderma atroviride, Sarocladium terricola, Arthrinium arundinis and Diaporthe eres) generally not associated with post-harvest fruit diseases. Species such as Penicillium ubiquetum, Cladosporium pseudocladosporioides, Lichtheimia ramosa, Sarocladium terricola, Diaporthe nobilis, Bipolaris secalis, Paraconiothyrium fuckelii and Galactomyces reessii that had never previously been isolated from grapevine or grape were also identified. Moreover, it was not possible to assign a species to some isolates, while some members of Didymosphaeriaceae and Didymellaceae remained unclassified even at genus level. This study provides insights into the diversity of the epiphytic fungi inhabiting withered grapes and evidences the importance of their identification to understand the causes of fruit diseases. Finally, phylogenetic species delimitation furnished data of interest to fungal taxonomy. •The mycobiota of withered grapes is characterized by extensive heterogeneity.•Phylogenetic analysis assigned taxon affiliation accurately to isolates.•Species generally not associated with post-harvest fruit diseases were identified.•Phylogenetic species delimitation furnished data of interest to fungal taxonomy.