The increased incidence of dental caries by cigarette smoking (CS) has been widely reported in epidemiological studies, but the relationship between CS and cariogenic biofilm growth has been rarely studied. This study aims to investigate the effects of CS exposure on the growth and virulence of Streptococcus mutans biofilms (S. mutans). Briefly, S. mutans biofilms were formed on saliva-coated hydroxyapatite disks, which were exposed to CS 1, 3, and 6 times per day, respectively. In addition, S. mutans biofilms without CS exposure were considered as the control group. Acidogenicity, dry weight, colony-forming units (CFUs), water-soluble/insoluble extracellular polysaccharides (EPSs), and intracellular polysaccharides (IPSs) were analyzed and confocal laser scanning microscopy (CLSM) images of 74-h-old S. mutans biofilms were obtained. The lowest accumulation of biofilms and EPSs were detected in the 6 times/day CS exposure group compared with those of the control group and other CS exposure groups in 74-h-old S. mutans biofilms. CLSM also revealed the lowest bacterial count (live and dead cells) and EPSs biovolume in the six times/day CS exposure group in 74-h-old S. mutans biofilms. CS exposure inhibited the growth of S. mutans biofilm in vitro study, the anti-cariogenic biofilm formation was enhanced with a dose (frequency)-dependent at which frequency has more influence in the present findings.