This study aimed to separate the effects of chemical composition and epiphytic microflora on the fermentation quality and bacterial community of Napier grass silage. Napier grass was harvested in two vegetative stages (Early, EG; Late, LG) with 24 or 28% dry matter, cut into theoretical lengths of 2–3 cm, and ensiled in bag-type silos containing 450 g fresh matter for 30 d. A 2 × 2 factorial arrangement of treatments (2 chemical composition × 2 epiphytic microbiota) in a completely randomized design were as follows: EG epiphytic microbiota + gamma-ray irradiated EG (EMEG), LG epiphytic microbiota + gamma-ray irradiated EG (LMEG), EG epiphytic microbiota + gamma-ray irradiated LG (EMLG), and LG epiphytic microbiota + gamma-ray irradiated LG (LMLG). Triplicates per treatment were sampled after 30 d of ensiling for fermentation quality and bacterial community analyses. The maturity of Napier grass had a great impact on its chemical composition and epiphytic microflora at harvest. Silages made from irradiated EG (EMEG and LMEG) had higher lactic acid concentration and lactic:acetic acid ratio, and lower pH value and ammonia nitrogen (NH3-N) concentration than the silages made from irradiated LG (EMLG and LMLG). Species differential analyses showed that the chemical composition variation significantly affected the relative abundance of Lactobacillus, Lactococcus, Enterobacter, Serratia, unclassified Lactobacillales, Leuconostoc and Pediococcus in EMEG and EMLG silages. While there was no effect of epiphytic microbial variation on the relative abundance of bacterial genera in EMLG and LMLG silages. These results manifested that, in this study, chemical composition rather than epiphytic microflora affected the succession of bacterial community in Napier grass silages, thereby affecting the final fermentation quality.