The aim of the study was to evaluate the antibacterial activity of essential oil extracted fromChrysanthemum boreale(C. boreale) onStreptococcus mutans(S. mutans). To investigate anticariogenic properties, and bacterial growth, acid production, biofilm formation, bacterial adherence ofS. mutanswere evaluated. Then gene expression of several virulence factors was also evaluated.C. borealeessential oil exhibited significant inhibition of bacterial growth, adherence capacity, and acid production ofS. mutansat concentrations 0.1–0.5 mg/mL and 0.25–0.5 mg/mL, respectively. The safranin staining and scanning electron microscopy results showed that the biofilm formation was also inhibited. The result of live/dead staining showed the bactericidal effect. Furthermore, real-time PCR analysis showed that the gene expression of some virulence factors such asgtfB,gtfC,gtfD,gbpB,spaP,brpA,relA, andvicR ofS. mutanswas significantly decreased in a dose dependent manner. In GC and GC-MS analysis, seventy-two compounds were identified in the oil, representing 85.42% of the total oil. The major components were camphor (20.89%),β-caryophyllene (5.71%),α-thujone (5.46%), piperitone (5.27%),epi-sesquiphellandrene (5.16%),α-pinene (4.97%), 1,8-cineole (4.52%),β-pinene (4.45%), and camphene (4.19%). These results suggest thatC. borealeessential oil may inhibit growth, adhesion, acid tolerance, and biofilm formation ofS. mutansthrough the partial inhibition of several of these virulence factors.