Streptococcus mutans are considered the most cariogenic bacteria, but it has been suggested that Candida albicans could increase their cariogenicity. However, the effect of this dual-species microorganisms’ combination on dentine caries has not been experimentally evaluated. Biofilms of C. albicans, S. mutans and C. albicans + S. mutans (n = 12/biofilm) were grown in ultra-filtered tryptone yeast extract broth culture medium for 96 h on root dentine slabs of known surface hardness and exposed 8 times per day for 3 min to 10% sucrose. The medium was changed 2 times per day (after the 8 cariogenic challenges and after the overnight period of famine), and aliquots were analyzed to determinate the pH (indicator of biofilm acidogenicity). After 96 h, the biofilms were collected to determine the wet weight, colony-forming units, and the amounts of extracellular polysaccharides (soluble and insoluble). Dentine demineralization was assessed by surface hardness loss (% SHL). The architecture of the biofilms was analyzed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Data were analyzed by ANOVA followed by Tukey’s test (α = 0.05). The dual-species C. albicans + S. mutans biofilm provoked higher % SHL on dentine (p < 0.05) than the S. mutans and C. albicans biofilm. This was supported by the results of biofilm acidogenicity and the amounts of soluble (6.4 ± 2.14 vs. 4.0 ± 0.94 and 1.9 ± 0.97, respectively) and insoluble extracellular polysaccharides (24.9 ± 9.22 vs. 18.9 ± 5.92 and 0.7 ± 0.48, respectively) (p < 0.05). The C. albicans biofilm alone presented low cariogenicity. The images by CLSM and TEM, respectively, suggest that the C. albicans + S. mutans biofilm is more voluminous than the S. mutans biofilm, and S. mutans cells interact with C. albicans throughout polysaccharides from the biofilm matrix. These findings show that C. albicans enhances the cariogenic potential of the S. mutans biofilm, increasing dentine demineralization Ver menos