The antifungal activity of Streptomyces griseorubens E44G against Rhizoctonia solani, the causal agent of root rot disease of corn, was investigated. The mycelial growth of R. solani was inhibited by S. griseorubens E44G, indicating that it has an antifungal potential. The antagonist, S. griseorubens E44G, was detected to have proteolytic activity, using the method of casein hydrolysis. Moreover, the protease production was optimized under submerged conditions. The purification and precipitation of protease were achieved by ammonium sulphate and Sephadex G-100 gel filtration chromatography. Protease activity was detected spectrophotometrically based on the production of tyrosine. The molecular weight of the enzyme (35 kDa) was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The optimum activity of the enzyme was detected at pH 8.5 and 60 °C. The results indicated that the enzyme was thermostable and retained full activity even after 1 hour of incubation at 60 °C. The purified enzyme substantially inhibited the growth of R. solani, indicating that this enzyme may be actually involved in the antagonistic process.