Background and Aims <p>Litter decomposition is a fundamental ecological process that regulates nutrient cycling, carbon sequestration, and soil fertility within ecosystems. This process is mediated by intricate interactions between biotic and abiotic factors, with microbial communities playing a central role in the decomposition of organic matter. However, mechanistic studies at the microbial community level in the process remain scarce. This study aims to fill this gap.</p> Methods <p>We conducted an incubation experiment involving two temperatures (15&#xa0;°C and 25&#xa0;°C), two soil types (forest and grassland), three litter types (broadleaf, coniferous and herbaceous). The microbial community composition under different treatments was investigated using high-throughput sequencing (16S and ITS) and analyzed by non-metric multidimensional scaling (NMDS) and permutational multivariate analysis of variance (PERMANOVA).</p> Results <p>Litter type emerged as the primary driver of microbial community structure. Herbaceous litter (C:N ratio = 20.9) exhibited a Cyanobacteria abundance exceeding 80%, leading to lower alpha diversity. As the C:N ratio increased, the absolute dominance of Cyanobacteria was limited, and oligotrophic microbes further differentiated, enhancing alpha diversity. While temperature did not exert a significant direct effect, higher temperatures amplified diversity differences among litter types, modulated by soil pH. Compared to bacteria, fungal communities were less responsive to these factors, potentially due to higher network modularity (0.698).</p> Conclusions <p>This study reveals how litter stoichiometry predominantly shapes microbial community composition during decomposition. These findings advance mechanistic understanding of litter decomposition and improving predictions of carbon and nutrient cycling dynamics under varying global environmental conditions.</p>