Development of new effective biocontrol agents is largely based on the antagonistic capacity of candidate agents against targeted pathogens in vitro. Different mechanisms contribute to such capacity, including the activity of cell wall-degrading enzymes, secretion of antimicrobial secondary metabolites, growth vigour and resistance to exogenous and endogenous toxins. In this study, a series of laboratory experiments were designed to improve the antagonistic activities of Trichoderma spp. against two plant fungal pathogens, Sclerotium rolfsii and Rhizoctonia solani. A simple but efficient mutagenesis programme was carried out using ultraviolet light to induce modifications in the genetic structure of two Trichoderma biocontrol agents, T. virens and T. asperellum. The obtained mutants were subjected to a) initial screening for media-permeable antifungal metabolites using the cellophane membrane-based method, and b) selected mutants were subjected to a series of antagonistic tests. Results revealed that the antagonistic potential of selected mutants was significantly improved against the two plant pathogens. Genetic stability test results indicated that the UV-derived mutant Tv3, maintained its elevated performance after 12 rounds of sub-culture. Gene expression analysis for five antagonism-associated genes were examined using real-Time PCR. Results revealed that the gene expression of two genes, chitinase 33, a cell wall degrading enzyme and, polyketide synthase, which is responsible for polyketide biosynthesis, a class of secondary metabolites with antimicrobial roles, were significantly upregulated in one of the mutated T. virens strains. Results of our in vitro antagonistic studies along with our molecular analysis indicate that the UV mutagenesis could be an effective strategy to improve Trichoderma antagonistic potential.