The use of fungi of the genus Trichoderma spp. for the control of plant diseases it has proved to be an important and promising tool, mainly for the tomato crop production system, however, there are difficulties in establishing the bioagent. This work aimed to evaluate the effect of substrate incubation time after inoculation with Trichoderma harzianum to control the damping-off of tomato seedlings. The experimental design was completely randomized, consisting of six treatments and four replications. The treatments are two incubation times in two doses of T. harzianum (0 and 10 days of substrate incubation [DIST] after inoculation with T. harzianum in 1.0 or 5.0 g of T. harzianum) and two controls (control inoculated and not inoculated with Rhizoctonia solani). The variables analyzed were incidence of damping-off, area under the disease progress curve, percentage of emergence, emergency speed index, average emergency time, germination speed coefficient, total fresh mass, root length (cm) and height of the area part (cm). The treatments containing Trichoderma harzianum have proven to be promising for the control of R. solani and for the growth of tomato seedlings.