Microplastics (MPs) transport bacteria from rivers to oceans, impacting aquatic ecosystems. In situ incubation, a common method for studying bacterial communities on MPs, cannot reproduce complex and realistic environmental dynamics. Herein, a natural incubation experiment was performed to reproduce the migration of nine different substrates from rivers to the sea and examine the succession of bacterial communities on their surfaces. Furthermore, an in situ sequential incubation experiment and the natural incubation experiment were compared. Substantial structural changes were observed in the bacterial communities on MPs along riverine courses to the ocean, with implications for biosecurity, elemental cycling, and degradation potential in aquatic ecosystems. Rapid fluctuations in salinity were the prevalent factor for the significant changes in the bacterial community on MPs. The type of substrate has a significant effect on the formation and structure of bacterial communities, which depend on substrate structure and composition. Unlike in situ incubation, natural incubation reproduces realistic environmental dynamics, providing more comprehensive information on bacterial species composition, diversity, functional metabolism, and structure on MPs. This study introduces a novel methodological approach for MP research and enhances our understanding of the ecological risks associated with MPs transported from rivers to the ocean.