Purpose: To test the hypothesis that the number of adhesive layers influences the adhesive interface properties under cariogenic challenge conditions using a Streptococcus mutans model. Materials and Methods: Bovine teeth (n = 90) were sectioned into blocks of 5 mm and divided into two groups for microleakage testing (n = 60) and tensile bond strength testing (n = 30). In each group, the samples were subdivided into subgroups according to the number of adhesive layers applied on the dentin: one (SB1), two (SB2), and three adhesive layers (SB3). The samples of the control groups were placed in BHI broth medium supplemented with 2% sucrose without microorganisms, and the experimental groups were submitted to Streptococcus mutans American Type Culture Collection (ATCC) for 5 days. For the tensile strength test, samples were sectioned into 1-mm-thick slices and submitted to a constant load of 0.5 mm/min in a universal testing machine. Fractured surfaces were analyzed and characterized as adhesive, cohesive, or mixed. The microleakage test was performed with silver nitrate solution. Results: In experimental groups, the tensile test revealed a statistically signifcant difference between the one- (18.59 ± 5.3) and three-layer (11.28 ± 5.0) groups (p 0.001; ANOVA and Tukey's test). The adhesive failure mode was slightly more frequent in the one- (60%) and three-layer (80%) adhesive application groups. On the other hand, the microleakage levels of all experimental groups were statistically similar (Kruskal-Wallis; p > 0.05). Conclusion: The experimental conditions influenced tensile properties and failure modes of different adhesive interfaces; however, they did not influence microleakage. Keywords: dentin bonding agent, tensile strength, dental leakage, Streptococcus mutans, dental caries