Bletilla striata (Thunb.) Rchb.f. is a traditional Chinese medicinal plant that is widely cultivated in China, planted across more than 10,000 ha in Guizhou. Anthracnose disease was recently observed to be widespread in these plantations of the Guizhou province. This study aimed to identify the causal agents of anthracnose targeting B. striata in the Guizhou Province. Pathogens isolated from symptomatic leaves were collected from the Zunyi and Xingyi cities of the Guizhou Province in 2022. We obtained 32 Colletotrichum isolates and divided them into four groups based on their morphological characteristics. Phylogenetic analysis of the rDNA internal transcribed spacer and the actin, chitin synthase, and glyceraldehyde-3-phosphate dehydrogenase gene sequences revealed four Colletotrichum species responsible for B. striata anthracnose in the Guizhou Province. These were identified as C. liriopes, C. fructicola, C. boninense, and C. plurivorum, accounting for 40.6%, 31.3%, 18.8%, and 9.4% of the incidence, respectively. The pathogenicity tests showed that four species of anthracnose fungi exerted variable pathogenic effects on B. striata. To the best of our knowledge, this is the first study that identifies C. liriopes, C. boninense, and C. plurivorum as etiological agents of B. striata anthracnose in Ghizhou, China.