OBJECTIVE: Urea ammoniation is one of the more effective ways for straw feed utilization. Current research on urea ammoniation has focused on chemical reactions in the process of ammoniating straw, neglecting the microbial-driven process. METHODS: This study aims to examine the effects of 2% and 4% urea on bacteria and fungi and their metabolites and fermentation quality of corn stover and rice straw under 40% and 60% moisture conditions. RESULTS: 4% urea ammoniation increased the total nitrogen content of corn stover and rice straw, and reduced the neutral detergent fiber and acid detergent lignin of rice straw. Lactic acid and acetic acid are produced during the urea ammonification process, and 2% urea treatment has the best promoting effect on it. 2% urea ammoniation also modified the composition of the Lactobacillales and increased the relative abundance of Enterococcus of corn stover and rice straw under 60% water, leading to changes in the main driving microbiota. Moreover, urea ammoniation can promote the metabolism of bacteria and fungi in degrading lignin, producing various lignin degradation products, such as vanillin, 4-hydroxybenzaldehyde, protocatechuic acid, sinapyl alcohol, benzaldehyde, benzoic acid, etc. CONCLUSION: Urea ammoniation is not only a chemical process, but also a microbial-driven process that involves changes in microbial composition and associated metabolism.