Trichoderma species have been investigated as biological control agents for over 70 years owing to their ability to antagonize plant pathogenic fungi. Mycoparasitism, one of the main mechanisms involved in the antagonistic activity of Trichoderma strains, depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. The antifungal activity of an alpha-1,3-glucanase (EC 3.2.1.59, enzymes able to degrade alpha-1,3-glucans and also named mutanases) has been described in T. harzianum and its role in mycoparasitic processes has been suggested. In this study, we report on the purification, characterization and cloning of an exo-alpha-1,3-glucanase, namely AGN13.2, from the antagonistic fungus T. asperellum T32. Expression at the transcription level in confrontation assays against the strawberry pathogen Botrytis cinerea strongly supports the role of AGN13.2 during the antagonistic action of T. asperellum.