The obtainment of 30 new strains from native Trichoderma harzianum after UV light irradiation (UV-A and UV-C), and of 82 strains resulted from protoplast fusion were accomplished. The new strains, initially selected for their growing rate under low temperature and high pH conditions, as well as for their innocuousness on tomato plants, were tested for in vitro inhibition of Pyrenochaeta lycopersici in dual cultures and due to secretion of volatile and diffusible metabolites. All the UV-A and UV-C selected candidate mutants were innocuous to tomato plants, but none of them showed improvement in their biocontrol activity on P. lycopersici. Th12A20.1 increased 1.3 and 1.9 fold the total fresh weight of Fortaleza tomato plants when compared to its parental strains Th12 and Th11, respectively. The selected candidate mutants obtained through protoplast fusion were also innocuous to tomato plants, but only ThF1-2 and ThF4-4 inhibited 1.3 fold (in dual cultures) and 5 fold (due to secretion of volatile metabolites) the growth of P. lycopersici, respectively, in relation to the mean inhibitory effect of both parents. Therefore, these candidate mutants could be included in experiments under field conditions.