Colonization of microorganisms in removable prosthesis is a recurrent problem, affecting almost the totality of prosthetic patients. In the present study, optical coherence tomography (OCT) was employed to evaluate in vitro the biofilm formation in complete dental prostheses. Four acrylic resins (Coe-Comfort, Soft-Confort, VipiCril Plus and Lucitone 550) were divided into four groups (n = 10), polymerized and polished according to their respective manufacturers’ guidelines. OCT scanning was performed for all samples before and 96 h after inoculation with Candida albicans and Staphylococcus aureus. The employed OCT system (Callisto SD-OCT, Thorlabs Inc.) operates in the Spectral Domain with central wavelength at 930 nm, 100 nm of spectral bandwidth, maximum output power of 5 mW, axial resolution of 7 / 5.3 μm in water / air, respectively, lateral resolution of 8 μm and depth of light penetration of 1.6 mm inside the sample. The biofilm evaluation was carried out in a qualitative/quantitative way through 2D optical images. The formed biofilm was clearly visualized and its thickness measured, besides observation of porosity according to the material employed. The results of the present study suggest that OCT is a viable technique to visualize the biofilm in all types of acrylic resin studied, besides allowing a view of the internal structure of the material and providing porosity data. It has the potential for clinical use in prostheses being used by patients, which are removed for evaluation.