Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, is one of the most important diseases of tomato crop in Brazil. Several alternatives to fungicides have been evaluated in search for products that control this disease and have low environmental impact and toxicity to living beings. In this context, the use of Trichoderma spp. is an alternative to agrochemicals, since they are bioregulators and antagonists of several phytopathogens. This work aimed to evaluate the antagonistic and mycoparasitory action of the Trichodermil 1306 biofungicide against F. oxysporum, and its action in the control of fusarium wilt in tomato seeds and seedlings. The biofungicide was used at concentrations of 0, 50, 100, and 150% of the dose recommended for application of 20 L with a backpack sprayer. The experiment was conducted in a completely randomized design with five replications. Data were submitted to analysis of variance and mean compared by Tukey’s test at 5% probability. The 100% Trichodermil concentration was the most efficient for treating tomato seeds. Although this concentration did not show differences in relation to the lowest dose used (50%) in the tomato seed antagonism, mycoparasitism and F. oxysporum incidence tests were recommended because that the lowest concentration resulted in 18% lower root protrusion than that observed with 100% dosage. The in vitro antagonistic and mycoparasitory action of this biological fungicide indicates its potential in the control of fusarium wilt in tomato crops.