Hydropower is a clean and renewable energy source, and cascade hydropower stations have been developed to enhance water energy utilization efficiency. While small hydropower stations have a smaller scale and environmental impact compared to large ones, the cumulative effects of cascade development on river ecosystems should not be overlooked. In this study, flow cytometry was used to classify picoplankton from water samples collected at four small cascade hydropower stations on a Pearl River tributary into six microbial groups: Virus, LNA (Low Nucleic Acid), HNA (High Nucleic Acid), Cyanobacteria, Algae, and Fungi. Four ecological assessment indices were calculated: Photosynthetic Autotrophic Capacity (PAC), Bacterial Activity Index (BAI), Virus Regulatory Capacity (VRC), and Fungal Metabolic Capacity (FMC). By analyzing trends in microbial abundance and ecological indices and their correlations with environmental factors, the results showed that along the small cascade hydropower stations, dissolved oxygen (DO) and electrical conductivity (EC) increased from 5.71 mg/L and 49.87 μS/cm upstream to 6.80 mg/L and 56.18 μS/cm downstream, respectively. In contrast, oxidation-reduction potential (ORP) and total organic carbon (TOC) concentrations decreased from 3.81 mV and 1.59 mg/L to −8.05 mV and 1.08 mg/L, respectively. Among the microbial groups, the abundance of Virus, LNA, and Fungi decreased by 30.9%, 30.5%, and 34.9%, respectively, along the cascade system. EC, TOC, and NO₃⁻-N were identified as key drivers of changes in the abundance of the Virus, LNA, and Fungi groups. The concentrations of carbon and nitrogen nutrients significantly influenced the ecological assessment indices. Cascade hydropower stations had a significant impact on PAC, BAI, and VRC, while their influence on FMC was relatively small. The VRC showed a decreasing trend, suggesting a weakening effect of the stations on VRC. This study offers new perspectives and methods that facilitate the rapid and quantitative assessment of the ecological impacts of cascade hydropower stations.