Determining the effects of fermentation duration on the microbial ecosystem, potential pathogenic risks, and metabolite generation during the fermentation of distilled grains is essential for safeguarding the safety and enhancing the nutritional profile of animal feed. This study investigates the effect of varying fermentation times (9, 30, and 60 days) on microbial diversity, pathogenic risk, and metabolite profiles in distiller grains using 16S rDNA sequencing and LC-MS-based metabolomics. The results showed that early fermentation (9-30 days) enhanced the abundance of beneficial bacteria, such as