Colletotrichum gloeosporioides is a pathogen that can be carried by chili seeds. Control usually uses synthetic fungicides, but this can have negative impacts on the environment, human health and resistance to pathogenic fungi. A more environmentally friendly and safe control alternative is to use a biofungicide made from antagonistic fungi isolated from the rhizosphere of Terong Asam (Solanum lapiocarpum Dunal). The research was carried out in vitro using dual culture method, namely testing the pathogen C. gloeosporioides against the antagonistic fungi Trichoderma hamatum, T. harzianum, T. asperellum, Aspergillus niger, A. flavus, A. terreus, and Penicillium sp. the rhizosphere of Terong Asam. The results showed that all fungi obtained could inhibit the growth of the C. gloeosporioides seven days after isolation with different percentages of inhibition. High category of antagonistic activity has in Penicillium sp. (68.53%), T. asperellum (67.77%), T. hamatum and T. harzianum (66.27%) each, while other species showed low antagonist activity, namely A. flavus (40.63%), A. niger (33.58%), and A. terreus (28.70%). The difference of the inhibitory power is determined by the activity of antagonistic fungal mechanisms controlling pathogens, namely competition for growth sites and nutrients, antibiosis compounds produced and mycoparasitism. Antagonistic fungi that have high potential activity are used as biofungicides seed treatment to control C. gloeosporioides which infects seeds.