Two xylanases were purified to electrophoretic homogeneity from the thermophilic fungus Sporotrichum thermophile grown in a submerged liquid culture using wheat straw as carbon source. The enzymes, StXyn1 and StXyn2, have molecular masses of 24 kDa and 48 kDa, respectively, and are optimally active at pH 5 and at 60 °C. Both enzymes displayed remarkable stability up to 50 °C for 1 h, exhibiting a half-life of 60 min (StXyn1) and 115 min (StXyn2) at 60 °C. Biochemical characterization of the two xylanases against poly- and oligosaccharides indicated that StXyn1 and StXyn2 hydrolytic profiles match those of xylanase family 11 and family 10, respectively. LC-MS/MS analysis provided peptide mass and sequence information that assisted the identification of the corresponding xylanase genes from the S. thermophile genome and the classification of the two purified StXyn1 and StXyn2 as a family GH11 and GH10 endo-1,4-β-xylanases, respectively.