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.