Chaetomium globosum Kunze, has been identified as a potential antagonist of Cochliobolus sativus (S. Ito & Kurib.) Deschler ex Dastur. (Syn = Drechslera sorokiniana). Production of antifungal compounds by Chaetomium globosum (Cg) and their role in suppression of spot blotch of wheat caused by this fungus under in vitro and in vivo has been evaluated. Interaction between Chaetomium globosum isolates and C. sativus showed mycoparasitism by isolates Cg 1 and Cg 6 whereas isolates Cg 2, Cg 3, Cg 4 and Cg 5 showed antibiosis. Syringe filtered culture extracts of Cg 2 completely inhibited mycelial growth of C. sativus in liquid broth. In vitro bioassays were undertaken by amending the medium with crude extracts and agar diffusion method in order to assess the fungistatic activity of crude extracts from culture filtrates of different isolates of Chaetomium globosum. Significant differences in antagonism between isolates were observed. Antifungal metabolite profiling, on TLC (Thin Layer Chromatography) plates identified 13 compounds in isolate Cg 2, 11 compounds in Cg 3 and 7 compounds in Cg 6. Isolate Cg 1 produced only two faint bands and Cg 5 produced two bands of the same Rf value but of higher intensity. The production of antifungal compounds by isolates was positively correlated with antagonism to C. sativus on seedlings in glasshouse studies. The results showed high antifungal metabolite production by isolate Cg 2, which also gave maximum bioefficacy under laboratory and glasshouse conditions.