Riverine bacterial communities are more shaped by species sorting in intensive urban and agricultural watersheds.

Bacterial communities play a crucial role in maintaining the stability of river ecosystems and driving biogeochemical cycling, exhibiting high sensitivity to environmental change. However, understanding the spatial scale effects and assembly mechanisms of riverine bacterial communities under distinct anthropogenic disturbances remains a challenge. Here, we investigated bacterial communities across three distinct watersheds [i.e., intensive urban (UW), intensive agricultural (AW), and natural (NW)] in both dry and wet seasons. We explored biogeographic patterns of bacterial communities and the influence of landscape patterns at multi-spatial scales and water chemistry on bacterial communities. Results showed that diversity was significantly lower in UW and AW compared to NW, particularly in the dry season. A gradient of diversity with NW > UW > AW was observed across both seasons ( < 0.05). Pseudomonadota, Bacteroidota, and Actinobacteriota were the most abundant phyla across all watersheds, with specific taxa enriched in each watershed (i.e., the class was significant enrichment in UW and AW, and in NW). The influence of landscape patterns on bacterial communities was significantly lower in human-disturbed watersheds, particularly in UW, where this influence also varied slightly from near riparian buffers to sub-watershed. Homogeneous selection and drift jointly dominated the bacterial community assembly across all watersheds, with homogeneous selection exhibiting a greater influence in UW and AW. Landscape patterns explained less variance in bacterial communities in UW and AW than in NW, and more variance was explained by water chemistry (particularly in UW). These suggest that the stronger influence of species sorting in UW and AW was driven by more allochthonous inputs of water chemistry (greater environmental stress). These findings provide a theoretical foundation for a deeper understanding of riverine bacterial community structure, spatial scale effects, and ecological management under different anthropogenic activities.