Hybrid Pennisetum, a top biomass energy source, faces usage limitations because of its scarce lactic acid bacteria and high fiber content. This study assessed the influence of rumen fluid pretreatment on hybrid Pennisetum’s silage, with focus on silage duration and rumen fluid effects on quality and fiber decomposition. Advanced third-generation sequencing was used to track microbial diversity changes and revealed that rumen fluid considerably enhanced dry matter, crude protein, and water-soluble carbohydrates, thus improving fermentation quality to satisfactory pH levels (3.40–3.67). Ideal results, including the highest fiber breakdown and enzymatic efficiency (47.23 %), were obtained with 5 % rumen fluid in 60 days. The addition of rumen fluid changed the dominant species, including Paucilactobacillus vaccinostercus (0.00 % vs. 18.21 %) and Lactiplantibacillus plantarum (21.03 % vs. 47.02 %), and no Enterobacter was detected in the high-concentration treatments. Moreover, strong correlations were found between specific lactic acid bacteria and fermentation indicators, revealing the potential of achieving efficient and economically beneficial hybrid Pennisetum production.