Although fluoride has been widely used as a preventive agent for dental caries, the effects of fluoride on the activities of biofilms in different stages of cariogenic biofilm formation are less studied. This study was designed to investigate the antibiofilm activity of sodium fluoride during the early and mature stages of Streptococcus mutans (S. mutans) biofilm formation. S. mutans biofilms were formed on saliva-coated hydroxyapatite disks. In the early (0-46 h) and mature (46-94 h) biofilm stages, the biofilms were treated with different concentrations of fluoride (250, 500, 1000, 2000 ppm; 5 times in total, 1 min/treatment). Acidogenicity, dry weight, colony-forming units (CFUs), water-soluble/insoluble extracellular polysaccharides (EPSs), and intracellular polysaccharides were analysed, and confocal laser scanning microscopy images were obtained of the two stages of biofilms to determine antibiofilm activities of fluoride at varying concentrations during the formation of early and mature biofilms. In the early stages of cariogenic biofilm formation, test groups with all fluoride concentrations significantly inhibited the growth of S. mutans biofilms. The antibiofilm and anti-EPS formation activities of the brief fluoride treatments increased with a concentration-dependent pattern. At the mature biofilm stage, only the 2000 ppm fluoride treatment group significantly inhibited biofilm accumulation, activity, and intracellular/extracellular polysaccharide content compared with those of the control and other fluoride treatment groups. The antimicrobial effect of fluoride treatment on the growth of S. mutans biofilms was linked with the stage of cariogenic biofilm formation. The inhibition of S. mutans biofilm growth by fluoride treatment was easier in the early formation stage than in the mature stage. Fluoride treatment in the early stage of cariogenic biofilm formation may be an effective approach to controlling cariogenic biofilm development and preventing dental caries.