Background: Anthracnose disease of chili caused by Colletotrichum spp is one of the most destructive diseases affecting chili fruits in Thailand and significantly reduces fruit quality and chili production. Currently, this disease is managed primarily with synthetic fungicides that may affect public health and the environment adversely. Consequently, there is a need for biological management options. In vitro and in vivo evaluations of the antagonistic activity of Talaromyces flavus Bodhi001, Talaromyces trachyspermus Bodhi002, Talaromyces flavus Bodhi003 and Neosartorya fischeri Bodhi004 against Colletotrichum capsici, the causal agent of chili anthracnose disease, were conducted in the present study. Methods: The activity of antagonistic fungi against C. capsici was determined using PDA plate by dual culture method. The spore suspensions of C. capsici and antagonistic fungi were prepared in sterile water and adjusted using a hemocytometer to achieve a final concentration of about 106 spores mL-1. Result: The most effective antagonistic strains were T. flavus Bodhi001 and N. fischeri Bodhi004, which inhibited the mycelial growth of C. capsici by 68.99% and 70.76%, respectively. Interestingly, the antagonistic T. flavus Bodhi001 strain was the most effective at reducing the severity of chili anthracnose in vivo by up to 80%. The biological control activity of T. flavus Bodhi001 was to produce antibiosis against C. capsici; therefore, testing can be recommended to confirm its field trial stability. The results indicate that the application of the antagonistic fungi T. flavus Bodhi001 may be quite effective in biological control of chili anthracnose.