The present study has been undertaken todetermine the efficacy of some antagonistic fungi isolated from the rhizosphereof wheat plants grown in Summer region, Diwaniya Governorate, Mid Iraq for thebiological control of Helminthosporium sativum, the causal organism ofroot rot disease of wheat in vitro. Three different species from thegenus Trichoderma: T. harzianum, T. pseudokoningii and T. lignorum,in addition to Stachybotrys atra and Penicillium sp. wereisolated from the rhizosphere of wheat plants. Laboratory experiments indicatedthat T. harzianum and S. atrawere highly antagonistic to the pathogen when grown together on potato dextroseagar in Petri plates. Microscopic examination of the mycelia showed that hyphaeof T. harzianum were parasitized H. sativum, coiled around itshyphae and caused its lysis, but did not penetrate inside the hyphae. However, S.atra was invaded colonies of H. sativum and caused severe hyphaldamage. In the experiments of culture filtrates of the antagonistic fungi, T.harzianum and S. atra, were able to suppress growth of H. sativum,if incorporated in the medium and proved to be effective in controlling thepathogen. Resultsof effect of filtrate on spore germination showed that about 80 and 95% of themare unable to germinate with high concentrations (15 or 20 %) of culturefiltrates of T. harzianum and S. atra respectively. However, theother antagonists: T. pseudokoningii, T. lignorum and Penicilliumsp. were less effective in inhibiting spore germination of the pathogen. Resultsof antagonistic effect of the culture filtrates on wheat seed infection, exceptT. lignorum, showed that seed colonization by the root rot fungus wasdecreased significantly at the concentrations of 15 and 20 % compared withcontrol. Seed coating with antagonistic fungi was the best biological seedtreatment for reducing seed rot and diseased seedling caused by H. sativum.Antagonistic fungi have no side effects on seed germination except Penicilliumsp. which reduced the seed activity and germination as compared with the otherantagonists and with the control. It can be concluded that T. harzianumis a strong mycoparasite and S. atra is a good antagonistic agent tocontrol H. sativum, but in fields may be their activity are conditionedby soil environment specially the microflora