Streptococcus mutans have destructive effects on the development of caries. It is mainly found in biofilm and saliva, and can cause caries activity. Silymarin is an extract from the dried seeds of the milk thistle and has antibacterial activity. Seemingly, silymarine extract can prevent the destructive effects of S. mutans. Thus, this study was conducted to evaluate the effects of silymarine extract against S. mutans virulence and caries development in rat model and in vitro condition.Antibacterial activity, glucan adherence and synthesis, biofilm formation and acid production were investigated under in vitro condition. Gene expressions of relA, gtfC, brpA and comDE were assessed. Dental caries development was also evaluated. The results showed decreased biofilm formation, glucan synthesis and adherence and decreased acid production in higher doses of silymarine (P<0.05). The results also showed decreased gene expressions of relA, gtfC, brpA and comDE in the animals treated with silymarine than the control group (P<0.05). A significant decrease in caries development was found in the intervention groups compared with the control group (P<0.05). It can be concluded that use of silymarine, as a supplement, can inhibit caries development. It is recommended that silymarin can be used as a commercial supplement and/or in combination with other agents to prevent the development of dental caries.