Mitochondria are essential for cellular functions across organisms. A proteomic approach was taken to separate and identify mitochondrial proteins from a strain of Trichoderma harzianum with well established biocontrol properties. We optimized a method for the preparation of a sample enriched with mitochondria by ensuring efficient cell lysis and including several washing steps to minimize cytoplasmic contamination. We separated hundreds of proteins, using two-dimensional gel electrophoresis and identified 31 protein spots from T. harzianum, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and liquid chromatrography with mass spectrometry. Over 50% of the identified proteins were known to localize in the mitochondria. Three protein spots were identified from T. harzianum and a further five protein spots from the genera Trichoderma and Hypocrea. The remaining protein spots were identified by cross-species identification from other filamentous fungi, including Neurospora crassa and Aspergillus spp, and yeasts, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. In total, we identified 25 protein spots from T. harzianum, representing proteins that have not been characterized in existing Trichoderma protein databases. To our knowledge, this is the first two-dimensional mitochondrial protein map of a filamentous fungus.