Aim To study the antifungal mechanism of proteases from Streptomyces phaeopurpureus strain ExPro138 towards Colletotrichum coccodes and to evaluate its utilization as biofungicide. Methods and Results We screened proteolytic Streptomyces strains from the yam rhizosphere with antifungal activity. Forty proteolytic Streptomyces were isolated, among which eleven isolates showed gelatinolytic activity and antagonistic activity on C.coccodes. Of the 11 isolates, protease preparation from an isolate designated ExPro138 showed antifungal activity. 16S rDNA sequence analysis of the strain showed 99% similarity with Streptomyces phaeopurepureus (EU841588.1). Zymography analysis of the ExPro138 culture filtrate revealed that the strain produced several extracellular proteases. The protease preparation inhibited spore germination, spore adhesion to polystyrene surface and appressorium formation. Microscopic study of the interaction between ExPro138 and C.coccodes revealed that ExPro138 was mycoparasitic on C.coccodes. The protease preparation also reduced anthracnose incidence on tomato fruits compared with untreated control. Conclusion This study demonstrates possibility of utilizing antifungal proteases derived from antagonistic microbes as biofungicide. Significance and Impact of the Study Microbial proteases having the ability to inhibit spore adhesion and appressorium formation could be used to suppress infection establishment by foliar fungal pathogens at the initial stages of the infection process.