Ambrosia beetles, including Platypus cylindrus, commonly act as vectors of Ophiostomatales. In an investigation of fungi associated with P. cylindrus, several species of Ophiostomatales were identified, including two species of Ophiostoma s. str. These two species were characterized based on comparisons of DNA sequences of the internal transcribed spacer regions including the 5.8S gene of rDNA and β-tubulin gene. Two distinct clades were discernable in the phylogenetic trees, supported by high bootstrap values. One clade included the species Ophiostoma tsotsi, reported herein for the first time in Tunisia, and the species Ophiostoma quercus.