In this in vitro study, cross-polarization optical coherence tomography (CP-OCT) was used to evaluate microleakage in dental composite restorations bonded to enamel with two types of one-step self-etching (SE) adhesives. One-step SE adhesives were proposed to simplify bonding of composite restorations. However, bonding of these simplified adhesive to dental enamel is still questionable. OCT is a promising diagnostic tool that allows micron-scale imaging of biological tissues and structures. Class-V cavities (4-mm diameter × 1-mm depth) were prepared on the labial surfaces of extracted human anterior teeth with margins and floor located in enamel. The cavities were then divided into two groups (n = 5) and restored with either Tetric N-Bond Universal (Ivoclar Vivadent, Liechtenstein) or Palfique Bond (Tokuyama Dental Corporation, Japan), followed by Estelite Flow Quick flowable composite (Tokuyama Dental Corporation). After storage in 100% humidity for 24 h, specimens were immersed in ammoniacal silver nitrate solution for another 24 h. Later, they were rinsed and immersed in photo-developing solution under fluorescent light for 8 h. CP-OCT at 1310 nm center wavelength was used to scan seven two-dimensional cross-sectional images from each specimen. Each cross-sectional image was analyzed using ImageJ software to quantify interfacial microleakage at the cavity floor. Student's t-test showed significant difference between the tested adhesives (p < 0.05). Palfique Bond adhesive presented inferior interfacial adaptation as it obtained higher values of floor microleakage compared to Tetric N-Bond Universal adhesive group. CP-OCT enabled nondestructive quantitative assessment of microleakage in enamel dental restorations.