Streptococcus mutans synthesizes 3 glucosyltransferases (Gtfs) associated with cariogenic biofilms, while commensal Streptococcus sanguinis produces only one; gtfP and hydrogen peroxide (H2O2) by SpxB. The aim was to test the hypothesis that under a sucrose-induced cariogenic challenge, the expression of competition-related genes is differentially regulated depending on whether S. sanguinis or S. mutans primarily colonize enamel. Dual-species biofilms of S. sanguinis and S. mutans were formed under different colonization sequences on enamel slabs and exposed to 10% sucrose for 5 min, 3×/day for 5 days. Biofilms were analyzed for the transcriptional response of competition-related genes encoding gtfB, gtfC, and gtfD for S. mutans and gtfP and spxB for S. sanguinis. In addition, acidogenicity (pH) and viable cells in each of the conditions were determined. For all the genes, a downregulation was observed during simultaneous colonization by both bacterial species. In contrast, gtfB was upregulated when S. sanguinis was the first colonizer (p < 0.05). Both gtfC and gtfD were upregulated during sequential inoculation with S. sanguinis as the first colonizer. An eleven-fold upregulation of gtfP was observed in biofilms with S. mutans as initial colonizer (p < 0.05), with a moderate increase in spxB expression. The lowest pH values and viable cells of S. sanguinis were observed when S. mutans first colonized the enamel slabs, compared to the other conditions (p < 0.05). Demanding sucrose-challenged oral environment requires increased expression of virulence traits to effectively compete and thrive in the dental biofilm, especially when the competitor has already colonized the ecological niche.