Abstract: In this study, Talaromyces flavus, a fungal antagonist, was isolated from soil samples that were collected from cotton fields in Golestan province of Iran. Antagonistic effects of non-volatile extracts of T. flavus isolates on Verticillium dahliae growth were investigated separately in laboratory experiments. Different isolates of T. flavus caused variable percents of growth inhibition of V. dahliae. Among isolates, ten of them that caused more growth inhibition of V. dahliae than other isolates, were selected for greenhouse experiments. In this step, first inoculum of microsclerotia of V. dahliae and seed treatments affected by non-volatile extracts and ascospores suspension of ten isolates of T. flavus were prepared. For comparison of infection index in seed treatment, greenhouse experiment was performed as a completely randomized design with forty-three treatments and three replications. Results of disease evaluation showed that among seed treatments affected by ascospores suspension of T. flavus isolates, seed treatments of isolates TF-2, Tf-12 and TF-17 caused maximum decrease in Verticillium wilt index. However, among seed treatments affected by non-volatile extracts of T. flavus isolates, seed treatments of isolates TF-1, TF-17 and TF-13 resulted in maximum decrease in disease index.