The antagonistic effect of the isolates of Gliocladium roseum, Saccharomyces cerevisiae, Sordaria fimicola, and their mixtures, at different concentrations against the cereal damping-off pathogen, Fusarium graminearum, was examined in vitro and in vivo (foliar, seed, soil, seed + soil) treatments on the susceptible wheat cultivars “Gun 91 and Sultan 95.” The 3 isolates inhibited growth of F. graminearum at a concentration of 1 × 109 spores/ml with inhibition rates of 84, 88, and 91%, respectively under in vitro conditions. For in vivo assays, the mixture of S. cerevisiae + S. fimicola exhibited a considerable antagonistic activity even at a concentration of 1 × 105 spores/ml. Particularly, at the seed + soil treatment of the mixture, the pathogen was almost completely suppressed with an inhibition rate above 96% at concentrations of 1 × 108 and 1 × 109 spores/ml for both wheat cultivars, and the percentage of emerged seedlings reached nearly 100%. The results verified that the mixture of S. cerevisiae + S. fimicola had a high potential, as a promising biocontrol agents and an eco-friendly alternative, to be used against the cereal damping-off caused by F. graminearum, to reduce the use of systemic fungicides.