The application of nanomaterials to contaminated river sediments could induce important changes in the speciation of heavy metals with potential impacts on ecosystem. Here, rhamnolipid (RL)-stabilized nanoscale zero-valent iron (RNZVI) was conducted to test its potential performance in changing the mobility and speciation of cadmium (Cd) in river sediments, with consideration of the influences of microbial community and organic carbon (OC). Compared to NZVI, RNZVI was more effective in transforming labile Cd to stable fraction with a maximum residual concentration increasing by 11.37 mg/kg after 42 days of incubation. Bacterial community structure was tracked using high-throughput sequencing of 16S rRNA genes. Results indicated that the application of RNZVI changed the bacterial community structure and increased the relative abundance of Fe(III)-reducing bacteria, which could redistribute Fe combined Cd into a more stable Fe mineral phase. The contents of OC were gradually decreased and became stable, might resulting from OC bioavailability's being stimulated by RNZVI through changing the bacterial community composition. This study indicates that abiotic process (i.e., from reaction with NZVI) and biotic process fueled by RNZVI lead to the immobilization of Cd in river sediments.