Chaetomium cupreum has a potential as biocontrol agent against a range of plant pathogens on the basis of production of antifungal metabolites, mycoparasitism, competition for space and nutrients, or various combinations of these. To explore genes expressed in C. cupreum, a cDNA library was constructed from mycelium and 3,066 expressed sequence tags (ESTs) were generated. Clusters analysis enabled the identification of 1,471 unigenes with 392 contigs and 1,079 singleton sequences. Putative functions were assigned to 874 unigenes that exhibited strong similarity to genes/ESTs in public databases putatively containing genes involved in cellular component, molecular function, and biological process. Other 597 ESTs representing novel genes showed no significant similarity to public database resource of NCBI. A proportion of genes was identified related to degradation of pathogen cell wall, antifungal metabolite production, as was estimated in the biocontrol fungus. The paper described is a first step towards the knowledge of the C. cupreum genome. The results present the useful application of EST analysis on C. cupreum and provide a preliminary indication of gene expression putatively involved in biocontrol.