Prunus belongs to the economically most important genera of fruit crops in Germany. Although wood pathogens possess the capability to damage the host substantially, the knowledge of the fungal pathogenic community and the mycobiome of Prunus wood in general is low. During a survey in important fruit production areas in Germany, branches with symptoms of fungal infection were sampled in Prunus avium, P. cerasus and P. domestica orchards, and 1018 fungal isolates were obtained primarily from the transition zone of symptomatic to non-symptomatic wood. By a combination of blastn searches and phylogenetic analyses based on ITS and LSU sequences with a strong focus on reliable reference data, a diversity of 172 fungal taxa belonging to Ascomycota, Basidiomycota and Mucoromycota were differentiated. The majority of the strains belonged to three classes of Ascomycota, namely Sordariomycetes, Leotiomycetes and Dothideomycetes. The dominant species were Aposphaeria corallinolutea (Dothideomycetes) and Pallidophorina paarla (Leotiomycetes) that were isolated more than a hundred times each, while all other taxa were isolated ≤ 30 times. Only part of them could be identified to species level. Because of the high plasticity of species boundaries, the identification certainty was divided into categories based on nucleotide differences to reference sequences. In total, 82 species were identified with high and 20 species with low (cf.) certainty. Moreover, about 70 species could not be assigned to a known species, which reveals Prunus wood to represent a habitat harbouring high numbers of potentially new species, even in a well-explored region like Germany.