Thielaviopsis basicola is a hemibiotroph fungus that causes black root rot disease in diverse plants with significant impact on cotton production in Australia. To elucidate how T. basicola growth and the proteome are influenced by interactions with natural sources, this fungus was cultured in the presence of root extracts from non-host (wheat, hairy vetch) and susceptible host (cotton, lupin) plants. We found that T. basicola growth was significantly favored in the presence of host extracts while hierarchical clustering analysis of two-dimensional electrophoresis protein profiles of T. basicola shows a dependency towards the plant rather than the host/non-host status. Analysis by LC-MS/MS of unique and differentially expressed spots and identification using tandem MS, cross-species similarity searching and de novo sequencing allowed successful identification of 41 spots. The identified proteins were principally involved in primary metabolism with smaller numbers implicated in other diverse functions. Identification of several “morpho” proteins suggested morphological differences that were further microscopically investigated. Identification of several highly expressed spots suggested that vitamin B6 is important in the T. basicola response to components present in hairy vetch extract, and finally, three spots, induced in the presence of lupin extract, may correspond to malic enzyme and be involved in lipid accumulation.