The present study is the first to reportXylaria karsticolaisolated from the basidiocarp ofMacrolepiota procera(Basidiomycota), from Stara Planina Mountain, Bulgaria and second report for such species found in Europe. The fungal isolate wasin vitrocultivated and the morphology was observed. It was primarily determined as a xylariaceous morphotype at the intragenus level, based on the evaluation of colony growth rate, color, and stromatic structure formation and was confirmed by unique conidiophores and conidia. The molecular identification of the isolate was performed by amplification of ITS1-5.8S-ITS2 region and the strain was identified asXylaria karsticolawith 97.57% of confidence. The obtained sequence was deposited in the GenBank database under the accession number MW996752 and in the National Bank of Industrial Microorganisms and Cell Cultures of Bulgaria under accession number NBIMCC 9097. The phylogenetic analysis of the isolate was also conducted by including 26 sequences obtained from differentXylariaisolates. Considering the phylogenetic data,X.karsticolaNBIMCC 9097 was grouped along with otherX.karsticolaisolates, although the DNA sequence of the novelX.karsticolawas rather distantly related to the otherX.karsticolasequence data. The results were supported by the bootstrap analysis (100%) and indicated the different origin of the examinedX.karsticolaNBIMCC 9097.