Trichoderma asperellum, a filamentous soil fungus, is an effective biocontrol agent against many fungal plant pathogenic species. In the present study, we investigated the biological control properties of the strain T. asperellum T4. T. asperellum fermentation products significantly decreased the ability of Rhizoctonia solani and Sclerotinia sclerotiorum to infect rice and soybean, respectively. To further elucidate the biocontrol mechanisms of T. asperellum at the molecular level, a cDNA library was constructed from its mycelium. In total, 3114 expressed sequence tags (ESTs) were generated, which represented 1,554 unigenes, including 354 contigs and 1,200 singletons. Among these unigenes, 731 represented known genes while 823 were novel genes. Forty-six unigenes potentially involved in biocontrol processes were identified from the EST collection. Among them, the expressions of 16 genes were studied, and 15 genes were highly differentially regulated during confrontation with 2 phytopathogens, suggesting that they play roles in the T. asperellum response to phytopathogens. Our study may provide helpful insight in the mechanism of biocontrol by T. asperellum T4 against plant pathogens.