River ecosystems are critical for human and environmental health, with bacterioplankton playing a vital role in biogeochemical cycles. Unveiling the spatial patterns of bacterioplankton communities in relation to environmental factors is important for understanding the processes of microbial variation and functional maintenance. However, our understanding of the correlations among bacterioplankton communities, physicochemical factors, and land use, especially in large rivers affected by intensive anthropogenic activities, remains relatively poor. Here, we investigated the bacterioplankton communities in July 2018 in three main tributaries of the Pearl River, i.e., Beijiang, Xijiang, and Pearl River Delta, based on 16S rRNA high-throughput sequencing. Results showed that the most dominant phyla, Proteobacteria, Actinobacteria, Cyanobacteria, and Planctomycetes accounted for 33.75%, 22.15%, 11.65%, and 10.48% of the total abundance, respectively. The bacterioplankton communities showed remarkable differences among the three tributaries in terms of composition, structure, diversity, and predictive functional profiles. Mantel and partial Mantel tests revealed that the bacterioplankton communities were affected by physicochemical variables (p < 0.01) and land use (p < 0.01). Redundancy analysis identified specific conductivity, dissolved oxygen, agricultural land, ammonium, urban land, and water transparency as the dominant environmental factors influencing the bacterioplankton communities in the Pearl River. Variation partitioning analysis indicated that both physicochemical factors and land use had direct effects on the bacterioplankton community, and that land use may also shape bacterioplankton communities through indirect effects of physicochemical factors on riverine ecosystems. This study provides fundamental information on the diversity, spatial patterns, and influencing factors of bacterioplankton communities in the Pearl River, which should enhance our understanding of how such communities change in response to environmental gradients and anthropogenic activities.