Streptococcus mutansform oral biofilms (BFs) and cause dental caries. Roasted green tea (RGT) is prepared by roasting the tea plant, and RGT-specific polyphenols are produced during the roasting process. Catechins, polyphenols in green tea, have BF inhibitory activity againstS. mutans; therefore, RGT-specific polyphenols are also expected to have this activity. However, there are few reports on the structural and functional properties of RGT. This study aimed to investigate the inhibitory activity of RGT againstS. mutansBF formation and to investigate the active compounds. RGT extract fractionation and BF inhibitory assay were performed. Strong activity was confirmed in the RGT fractions that had medium-high hydrophobicity, were rich in phenolic hydroxyl groups, and lacked catechins. A peak comprising compounds with molecular weights of 918 (mw918) and 1050 (mw1050) was purified from the fraction. Since BF inhibitory activity was confirmed for this peak, these compounds were considered to be part of the active ingredients. The mw918 polyphenol was detected only in RGT and it was thought to be produced during the roasting process. The results of this research will serve as a basis for the future application of RGT as a safe and effective anti-caries agent.