Background <p>Understanding the impact of nitrogen (N) addition on microbial interactions and community assembly during straw decomposition is essential for optimizing fertilizer utilization and straw returning in agroecosystems. Despite extensive research on the effects of N fertilization on microbial community diversity, there is limited information available regarding inter-kingdom interactions and community assembly during straw decomposition.</p> Methods <p>We conducted a four-month field experiment to investigate straw decomposition under different levels of N addition in winter. Microbial community diversity, inter-kingdom interactions and assembly processes in the straw residues was analyzed using high-throughput sequencing, co-occurrence networks, and ecological null modeling.</p> Results <p>Both α-diversity and β-diversity of microbial communities associated with straw residues increased, accompanied by significant dynamic shifts in community composition and structure across different stages of decomposition, particularly under conditions with a higher C/N ratio. The straw with the highest C/N ratio (40.46) exhibited the greatest decomposition ratio (19.3%) and microbial community dissimilarities over time (bacteria: r² = 0.705, <i>p</i> &lt; 0.001; fungi: r² = 0.636, <i>p</i> &lt; 0.001). Proteobacteria, Bacteroidota, Actinobacteriota, and Ascomycota were dominant decomposers. As decomposition progressed, the inter-kingdom interaction networks transitioned from complex to simpler, yet exhibited a higher frequency of interactions between Spirochaetota, Fibrobacterota, Myxococcota, Acidobacteriota, and Ascomycota. Bacterial community was predominantly influenced by deterministic processes, while fungal community assembly became increasingly stochastic.</p> Conclusion <p>These findings highlight the importance of microbial interactions and community assembly in straw decomposition, offering insights for optimizing straw management and N fertilization in sustainable agriculture.</p>