Rhizomania is one of the most important diseases of sugar beet around the world – including in Iran. The disease causes a severe decrease in sugar yield and is a limiting factor in sugar beet cultivation. Control of the disease is very difficult due to the long- term survival of its fungal vector (Polymyxa betae) in the soil. In this study, we investigated the effects of antagonistic fungal isolates on the population of the resting structure (cystosorus) of P. betae, under greenhouse conditions. Antagonistic fungi, including Trichoderma harzianum and Talaromyces flavus, were isolated from soil samples collected from sugar beet infested fields in the Semnan Province of Iran. In the next step, their inocula were prepared through reproduction on rice bran. For evaluation of the efficacy of antagonists in greenhouse conditions, a split plot trial was conducted and performed. The main factor was three different methods of application of T. flavus as the soil treatment, seed treatment, and a combination of both methods. The subfactor was the use of different fungal isolates. To determine the cystosorus population of the fungal vector, seedling roots in all treatments were stained with lactic acid and fuchsine (lactofushine), 60 days after sowing. The number of cystosorus in one gram of root was counted using a light microscope and hemocytometer. At the end of the study, average root weight in different treatments was also measured to select and introduce the best treatments in regard to their effects on root weight. According to the results, the number of cystosorus in 1 g of root was different in various treatments and those treatments containing TF-Su-M-1, TF-Su-M-2, TH-Su-M-1, and TH-Su-M-2 used as a soil application method were more effective in the reduction of the cystosorus population and root weight increase. Among the above-mentioned treatments, maximum reduction of cystosori population and the increase in root weight were observed in TH-Su-M-1 and TF-Su-M-2 through the soil application method.