There are signs that amyloid fibers exist in Streptococcus mutans biofilm recently. However, the characteristics of amyloid fibers and fibrillation influencing factors are unknown. In this study, we firstly used transmission electron microscopy (TEM) and atomic force microscopy (AFM) to observe the morphology of amyloid fibers in S. mutans . Then the extracted amyloid fibers from biofilm were studied for their characteristics. Further, the influencing factors, PH, temperature and eDNA, were investigated. Results showed there were mainly two morphologies of amyloid fibers in S. mutans , different in width. Amyloid fibers inhibitor-EGCG obviously destroyed biofilm at different stages, which is dose-dependent. The amount of amyloid fibers positively correlated with biofilm biomass in clinical isolates. Acidic pH and high temperature obviously accelerated amyloid fibrillation. During amyloid fibrillation, amyloid growth morphologies were observed by TEM and results showed two growth morphologies. Amyloid fibers formed complex with eDNA, which we call (a)eDNA. The molecular weight of (a)eDNA was similar to genomic DNA, greatly larger than that of eDNA in matrix. Combined use of DNase I and EGCG was more efficiently in inhibiting amyloid fibers and biofilm biomass. In conclusion, amyloid fibers are the crucial structures for S. mutans biofilm formation, showing two types of morphology. Acidic pH and temperature can obviously accelerate amyloid fibrillation. Amyloid fibers form complex with (a)eDNA and combined use of DNase and amyloid fiber inhibitor is more efficiently in inhibiting S. mutans biofilm formation.