Nicotine has well-documented effects on the growth and colonization of Streptococcus mutans. This study attempts to investigate the effects of nicotine on pathogenic factors of S. mutans, such as the effect on biofilm formation and viability, expression of pathogenic genes, and metabolites of S. mutans. The results demonstrated that addition of nicotine did not significantly influence the viability of S. mutans cells. The biofilms became increasingly compact as the concentrations of nicotine increased. The expression of virulence genes, such as ldh and phosphotransferase system (PTS)-associated genes, was upregulated, and nlmC was upregulated significantly, while ftf was downregulated. The lactate concentration of S. mutans grown in 1 mg/mL of nicotine was increased up to twofold over either biofilm or planktonic cells grown without nicotine. Changes in the metabolites involved in central carbon metabolism from sucrose indicated that most selected metabolites were detectable and influenced by increased concentrations of nicotine. This study demonstrated that nicotine can influence the pathogenicity of S. mutans and may lead to increased dental caries through the production of more lactate and the upregulation of virulence genes.