As changes in hard or soft oral tissues normally have a microbiological component, it is important to develop diagnostic techniques that support clinical evaluation, without destroying microbiological formation. The optical coherence tomography (OCT) represents an alternative to analyze tissues and microorganisms without the need for processing. This imaging technique could be defined as a fast, real-time, in situ, and non-destructive method. Thus, this study proposed the use of the OCT to visualize biofilm by Candida albicans in reline resins for removable prostheses. Three reline resins (Silagum-Comfort, Coe-Comfort, and Soft-Confort), with distinct characteristics related to water sorption and fungal inhibition were used. A total of 30 samples (10 for each resin group) were subjected to OCT scanning before and 96 h after inoculation with Candida albicans (URM 6547). The biofilm analysis was carried out through a 2D optical Callisto SD-OCT (930 nm) operated in the spectral domain. Then, the images were preprocessed using a 3×3 Gaussian filter to remove the noise, and then Otsu binarization, allowing segmentation and pixel counting. The layer’s biofilm formed was clearly defined and, indeed, its visualization is modified by water sorption of each material. Silagum-Comfort and Soft-Confort showed some similarities in the scattering of light between the clean and inoculated samples, in which, the latter samples presented higher values of light signal intensity. Coe-Comfort samples were the only ones that showed no differences between the clean or inoculated images. Therefore, the results of this study suggest that OCT is a viable technique to visualize the biofilm in reline materials. Because findings in the literature are still scarcely using the OCT technique to visualize biofilm in reline resins, further studies are encouraged. It should not contain any references or displayed equations.