Background <p>Micro-ecological islands provide unique habitats for microbes and play a crucial role in the functioning of aquatic ecosystems. Microbes settle on these micro-ecological islands, forming distinct microbial communities. Previous studies have provided some understanding of the colonization processes and regulatory mechanisms of protozoa in microbial communities. However, these islands are also subject to colonization by a variety of microbes beyond protozoa, and comprehensive cross-kingdom studies and their potential mechanisms remain largely unexplored.</p> Results <p>Using polyurethane foam units (PFU) to simulate micro-ecological islands, we studied the colonization dynamics of microbes in two distinct aquatic ecosystems, the Yangtze River and East Lake. Over 10-day colonization survey was conducted, we applied eDNA-PFU technology combined with metagenomic sequencing to comprehensively identify species present in the microbial communities, including bacteria, fungi, flagellates, protozoa, and metazoa. We found that microeukaryotes, rather than prokaryotes, were the primary colonizers in these two aquatic ecosystems. Our study reveals a colonization process of microeukaryotes in PFUs, profoundly influenced by their motility modes. Additionally, we propose a hypothetical food web framework within micro-ecological islands that maintains community stability, representing the most fundamental biological interactions.</p> Conclusions <p>Overall, this study enriches our understanding of micro-ecological islands and provides deeper insights into the colonization processes and regulatory mechanisms of microbial communities. It highlights the practical significance of micro-ecological islands in biological resource management, environmental protection, and biodiversity conservation.</p>