Objective This study aimed to evaluate the effects of synthetic antimicrobial decapeptide on the growth and structure of Streptococcus mutans biofilm. Methods In our previous study, the antimicrobial activity of decapeptide (KKVVFKVKFK-NH2) against major cariogenic and periodontopathogenic bacteria and Candida albicans was investigated in vitro. Streptococcus mutans, the causative agent of dental caries, was chosen for in-depth evaluation. In the present study, the antimicrobial activity of decapeptide against the growth and structure of Streptococcus mutans biofilm was investigated by determining minimum biofilm inhibition concentration(MBIC) and minimum biofilm reduction concentration (MBRC). The morphological characteristics and activity of bacterial biofilm were observed through confocal laser scanning microscopy(CLSM). Results Decapeptide suppressed Streptococcus mutans biofilm formation at MBIC of 62.5 μg?mL-1 to 125 μg?mL-1; decapeptide also reduced microbial activity of Streptococcus mutans biofilm developed for 24 h at MBRC of 250 μg?mL-1 to 500 μg?mL-1. CLSM results revealed that the cells in decapeptidetreated groups were disrupted; cell debris was also observed in decapeptide-treated groups. Conclusion This study indicated that the effect of decapeptide on bacterial biofilm not only inhibited bacterial biofilm growth but also disrupted mature biofilm structure and composition that developed for 24 h.