Fungi are renowned for their ability to produce extracellular enzymes into their surrounding environment. Xylanases are hydrolytic enzymes capable of xylan degradation. The objectives of this study were to isolate, screen for potential xylanolytic fungi from soil and tree bark samples from three locations in South Africa and to determine their growth conditions for maximum xylanase production. Forty-six isolates were obtained based on clearing zone formation on xylan-enriched agar plates using Congo red indicator. Xylanase activity was quantified during submerged fermentation. Isolate MS5, identified as Trichoderma harzianum with the highest enzyme activity (38.17 U/ml) was selected for further studies based on thermophilic properties (70℃) and pH (5.0). The culture conditions; incubation period (5 days), agitation speed (160 rpm) wheat bran (1%) and ammonium sulphate (1.2%) were optimised further. Biochemical characterisation of the crude enzyme revealed two pH and temperature optima (pH 6.0 at 60℃ and 70℃, pH 8.0 at 55℃ and 75℃). The enzyme retained >70% activity after 4 h at pH 6.0 at 70℃. SDS-PAGE revealed multiple protein bands with a prominent band at 70 kDa. Substrate Native PAGE revealed multiple isoforms between 55 and 130 kDa. This enzyme will be beneficial for applications in the animal feed and biofuels industries.