Luffa sponge gourd (Luffa cylindrica) is an important cucurbitaceous vegetable and is known as the source of loofah. From 2020 to 2021, a leaf disease occurred on luffa leaves in the Hunan Province of China. Symptoms were displayed as oval to irregular chlorotic lesions surrounded by yellow halos. The pathogens were isolated from the affected leaves. According to morphological characterization and molecular identification using multi-locus phylogenetic analysis of the internal transcribed spacer (ITS), actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB2), and partial mating type (Mat1-2) gene (ApMAT) regions, the pathogens were identified as two Colletotrichum species: Colletotrichum fructicola and C. siamense. Koch’s postulates were identified by a pathogenicity test and re-confirmation. To the best of our knowledge, C. fructicola and C. siamense are two new species associated with luffa sponge gourd anthracnose.