During a survey investigating fungal diversity in the Chishui River Basin, Guizhou Province, China, four fungal specimens were collected from submerged wood in freshwater habitats. Based on detailed morphological observations and multi-gene phylogenetic analyses, two novel species, Chloridium chishuiensis and Xylolentia chishuiensis, are herein described and illustrated in detail. Chloridium chishuiensis is phylogenetically closely related to C. crousii, C. kirkii, C. tropicale, and C. xishuangbannaense but differs from these taxa by producing multiple conidiogenous loci and possessing longer conidiophores. Xylolentia chishuiensis forms a sister lineage to X. oblongispora and is distinguished by its smaller, ellipsoid to subglobose conidia. Additionally, a new host record of Rhamphoriopsis aquimicrospora is reported, accompanied by a new geographical record of Xylolentia yibinensis. These findings enriched the fungal diversity of the Chishui River Basin.