Six isolates of Trichoderma spp. (belonging to species; Trichoderma harzianum and T. longibrachiatum) were applied as seed or soil treatments to suppress damping-off of seedlings of ten cotton cultivars under greenhouse conditions. In most cases, cultivar x isolate interaction was a highly significant (p < 0.01) source of variation in the tested seedling growth parameters: incidence of disease, seedling height, and seedling dry weight. This interaction implies that a single isolate of Trichoderma can be highly effective in controlling the disease on a cotton cultivar but may have minimal efficiency in controlling the disease on another cultivar. It was also found that, in most cases, cultivar x isolate x application method was a highly significant source of variation (p < 0.01) in the tested growth parameters. Cotton cultivars showed differences in the disease reaction to the biocontrol agents. In the experiments evaluating the Trichoderma antagonists and their effect on seedling disease, a highly significant (p < 0.01) experimental treatment interaction was found. This interaction suggests that the outcome of cultivar x isolate interaction is markedly affected by the application method. Thus, the application method should be chosen to maximize the outcome of this interaction. The degree of the control of seedling disease in cotton differed according to the isolates of antagonists, the application method and cultivars.