AIMS: There is scarce information on the occurrence of several fungi that infect withered grapes to produce passito wine. Isolation and characterization of Neofusicoccum parvum strains and evaluation of their effects on withered grape and wine were carried out. METHODS AND RESULTS: Nine isolates were phenotypically characterized by colony morphology and genetically discriminated by molecular methods. Two representative strains were identified as N. parvum according to the phylogenetic analysis of internal transcribed spacer (ITS), and a part of translation elongation factor 1‐alfa (TEF) and β‐tubulin DNA sequences. The pathogenicity of both strains on grape berries varied according to the inoculation and incubation conditions. Under withering conditions, infected berries showed browning and shrivelling and some berries showed pycnidial development on the surface. The infection affected laccase, esterase, β‐glucosidase and tannase on grape juice as well as the content of several aroma molecules on resulting wines. Strain‐specific effects on wine composition were also observed. CONCLUSIONS: Neofusicoccum parvum occurred in withered grapes and was able to infect grapes under withering condition changing the aroma wine. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports for the first time the N. parvum isolation in fruit‐drying rooms and indicates its important role on postharvest grape infection.