Background/Objectives: Dental biofilm formation on prosthetic materials contributes to oral diseases and material degradation. This pilot study evaluated the feasibility of combining time-domain optical coherence tomography (TD-OCT) and non-contact atomic force microscopy (NC-AFM) to characterize biofilm-associated optical and surface changes in dental prosthesis materials following antiseptic treatment. Methods: Acrylic resin, composite resin, and stainless-steel specimens were inoculated with Streptococcus mutans biofilms and exposed to water, Orahex, and Watsons mouthwash. TD-OCT was used to obtain extinction coefficient (EC) and optical thickness measurements, while NC-AFM was performed on Orahex-treated specimens to assess nanoscale surface morphology. EC distributions were analyzed descriptively from valid TD-OCT A-scans. Results: Within the examined specimens, EC distributions varied following biofilm formation and antiseptic treatment, with material-dependent responses. Composite resin exhibited the clearest reduction in EC following Watsons treatment, whereas acrylic resin and stainless-steel specimens showed more variable responses. Physical thickness remained relatively stable, while NC-AFM observations of the Orahex-treated specimens supported the TD-OCT findings by demonstrating corresponding surface morphological changes. Conclusions: This pilot study demonstrates the preliminary feasibility of integrating TD-OCT with NC-AFM for non-destructive evaluation of biofilm-associated optical and surface changes in dental prosthesis materials. The findings are descriptive and exploratory, highlighting the potential of TD-OCT for monitoring prosthetic materials while emphasizing the need for future studies incorporating independent biological replicates and complementary biological validation techniques.