Microplastics as a carrier can promote microbial diffusion, potentially influencing the ecological functions of microbial communities in aquatic environments. However, our understanding of the assembly mechanism of microbial communities on different microplastic polymers in freshwater lakes during succession is still insufficient, especially for the eukaryotes. Here, the colonization time, site, and polymer types of microplastics were comprehensively considered to investigate the composition and assembly of prokaryotic and eukaryotic communities and their driving factors during the lacustrine plastisphere formation. Results showed that the particleassociated microorganisms in water were the main source of the plastisphere prokaryotes, while the free-living microorganisms in water mainly accounted for the plastisphere eukaryotes. The response of prokaryotic communities to different microplastic polymers was stronger than eukaryotic communities. The assembly of plastisphere prokaryotic communities was dominated by homogenizing processes (mainly homogenous selection), while the assembly of eukaryotic communities was dominated by differentiating processes (mainly dispersal limitation). Colonization time was an important factor affecting the composition of prokaryotic and eukaryotic communities during the formation of the plastisphere. The Chao1 richness of prokaryotic communities in the plastisphere increased with the increase of colonization time, whereas the opposite was true in eukaryotic communities. This differential response of species diversity and composition of prokaryotic and eukaryotic communities in the plastisphere during dynamic succession could lead to their distinct assembly processes. Overall, the results suggest that distinct assembly of microbial communities in the plastisphere may depend more on specific microbial sub-communities and colonization time than polymer types and colonization site.