The aim of this study was to investigate the difference in composition and virulence of Streptococcus mutans biofilms according to substratum direction. S. mutans biofilms (46-h-old) were formed on three different saliva-coated hydroxyapatite (sHA) disc direction groups: downward (discs placed in the direction of gravity), vertical (discs placed parallel to gravity direction), and upward (discs placed opposite to gravity). The 46-h-old biofilms on sHA discs in the upward direction showed the highest biofilm accumulation, colony forming unit (CFU) count, and extracellular polysaccharide (EPS) amount, followed by those in the vertical and downward directions. In the confocal laser scanning microscopy (CLSM) study, the biofilms in the upward direction also showed the highest bacterial count (live or dead cells) and EPS biovolume. Scanning electron microscopy (SEM) analysis confirmed the microbiological and biochemical results. In addition, biofilm density and acid production were higher in the upward direction than those in the other directions. Our findings suggest that substratum direction, which might be related to gravity, strongly influences the formation and virulence of cariogenic biofilms and subsequent initiation of dental caries. Collectively, the differences in the formation and virulence of cariogenic biofilms are related to the direction of tooth surface (occlusal surfaces of mandibular teeth > proximal surfaces > occlusal surfaces of maxillary teeth).