BACKGROUND/PURPOSE: With advancements in digital technology, fully digital workflow for complete denture fabrication using 3D-printed denture base resin (DBR) has gained increasing clinical acceptance in recent years. However, the surface characteristics, biocompatibility, and biofilm formation of 3D-printed DBR materials remain insufficiently understood. Therefore, in this study, we investigated and analyzed these aspects. MATERIALS AND METHODS: Disk-shaped DBR specimens (Ø 2.5 mm, 3 mm thick) were fabricated using packed (PA), milled (ML), and 3D-printed (3D) processes. All specimens were ground with silicon carbide sandpaper (#600) and ultrasonically cleaned. Surface microtopography and sub-micron roughness were analyzed using scanning electron microscopy and atomic force microscopy, while a goniometer was used to measure contact angles to calculate the surface energy. Human gingival fibroblasts and were cultured on the specimens to assess the cytotoxicity and biofilm formation. Statistical analyses were performed with a significance level set to 0.05. RESULTS: Microscopic imaging revealed that the 3D group exhibited a more uniformly distributed texture, while it also had the lowest surface roughness (0.85 μm). Additionally, the PA group had the most hydrophobic surface (82.47°) and the highest surface free energy (46.08 mN/m). Notably, no group showed cytotoxic effects after 72 h of testing. In addition, the 3D group demonstrated the lowest biofilm formation after both 24 h and 72 h of microbial culture. CONCLUSION: 3D-printed DBRs exhibited the lowest surface roughness, maintaining non-cytotoxic and superior resistance to microbial adhesion, suggesting their potential for complete denture fabrication, easy maintenance of oral hygiene, and long-term clinical performance.