Optical coherence tomography (OCT) is a real-time, in-situ, non-invasive imaging device that is able to perform a cross-sectional evaluation of tissue microstructure based on the specific intensity of back-scattered and reflected light. The aim of the present study was to define normal values of epithelial thickness within the oral cavity. OCT measurements of epithelial thickness were performed in 28 healthy patients at six different locations within the oral cavity. Image analysis was performed using Image J 1.52 software. The healthy epithelium has a mean thickness of 335.59 ± 150.73 µm. According to its location within the oral cavity, the epithelium showed highest values in the region of the buccal mucosa (659.79 µm) and the thinnest one was observed in the mouth’s floor (100.07 µm). OCT has been shown to be useful for the evaluation of oral mucosa in vivo and in real time. Our study provides reference values for the epithelial thickness of multiple sites within the oral cavity. Knowledge of the thickness values of healthy mucosa is, therefore, of fundamental importance.