Recently, more and more new fungal pathogens have been described as causal agents of grapevine trunk diseases (GTDs), which lead to increasingly significant economic losses in viticulture worldwide. The genus Neofabraea consists of species mainly known as important plant pathogens causing perennial canker and bull's eye rot, a common postharvest disease of apple (Malus domestica) and pear (Pyrus communis) fruits. Neofabraea kienholzii also causes lesions on pome fruits and canker on woody tissues, but its pathogenicity has not been demonstrated on grapevine yet. In 2015, two strains, identified as N. kienholzii based on ITS sequence data, were isolated from vines showing symptoms of GTDs in Hungary. For an unambiguous taxonomic placement of the isolates, four loci (ITS, LSU, TUB2 and RPB2) were amplified and sequenced. The phylogenetic analysis confirmed that the two isolates represent N. kienholzii. Pathogenicity tests performed on potted grapevines, shoots, and canes confirmed the virulence of these fungi. Their growth and sporulation on different media were also investigated. To the best of our knowledge, this is the first proof of N. kienholzii might cause symptoms on Vitis and might have a role in GTDs.