Headwater streams, located upstream of the river network, play a crucial role in river ecosystems. However, the increasing conversion of natural habitats into agricultural land leads to extensive alterations in landscape composition, posing serious threats to natural stream ecosystems and biodiversity. This research focuses on the headwater streams of the Xin’an River in the Wannan Mountains, China. The study merged information on habitat conditions from two types of habitats (disturbed and natural segments) to evaluate the impact of agricultural land use on the beta diversity of fish assemblages. The results indicated that the disturbed segments exhibited intermediate land-use intensities, slightly higher habitat heterogeneities, and a higher number of native invasive species compared to the natural segments. Differences in local-scale habitat conditions and regional-scale land use drove the species composition discrepancy (taxonomic beta diversity) between disturbed and natural segments. This study provides evidence of how agricultural land use impacts fish assemblage diversity in headwater streams and underscores the significance of sustainable management and conservation for preserving endemic assemblages in these streams.