Purpose: To evaluate the effects of multiple adhesive layers of three etch-and-rinse adhesives on both adhesive thickness and microtensile bond strength (µTBS). Materials and Methods: Midcoronal occlusal dentin of 36 extracted human molars was used. Teeth were randomly assigned to 3 groups (EB, XP, PQ) according to the adhesive system to be used: PQ1 (Ultradent) (PQ), EnaBond (Micerium) (EB), or XP Bond (Dentsply/DeTrey) (XP). Specimens from each group were further divided into three subgroups according to the number of adhesive coatings (1, 2, or 3). In all subgroups, each adhesive layer was light cured before application of each additional layer. After bonding procedures, composite crowns were incrementally built up. Specimens were sectioned perpendicular to the adhesive interface to produce multiple beams, approximately 1 mm2 in area. Beams were tested under tension at a crosshead speed of 0.5 mm/min until failure. Adhesive thicknesses and failure modes were evaluated with SEM. The µTBS data and mean adhesive thickness were analyzed by two-way ANOVA and multiple-comparison Tukey's test (α = 0.05). Results: The mean bond strength (in MPa (SD)) of group EB gradually increased from 1 to 3 consecutive coatings (27.02 (9.38) to 44.32 (4.93), respectively) (p 0.05). The highest mean bond strengths for the PQ (46.66 (12.95)) and XP groups (40.55 (5.69)) were obtained applying two adhesive coatings. The mean thickness of the adhesive layer (in µm (SD)) significantly increased with the number of coatings (p 0.05), ranging from 29.45 (1.42) to 77.64 (1.10) for PQ, from 5.12 (0.68) to 37.75 (0.92) for EB, and from 12.64 (0.68) to 37.92 (0.71) for the XP group. Failure modes for EB specimens were mainly classified as adhesive failure between adhesive and dentin. The XP3 and PQ3 subgroups showed a greater number of total cohesive failure in adhesive. Conclusion: Multiple adhesive coats significantly affected bond strength to dentin. An excess of adhesive layer thickness can negatively influence the strength and the quality of adhesion. Keywords: dentin adhesive, adhesive layer, microtensile bond strength, SEM