Background: Monolithic zirconia has attracted considerable interest in dentistry due to its favorable physical and mechanical properties, making it a promising alternative for crown fabrication. Nonetheless, a standardized finishing protocol for this material has yet to be established. Objective: This study aimed to evaluate the surface characteristics and in vitro biofilm formation of zirconia finished by either polishing or glazing. Methods: A total of 72 zirconia specimens were fabricated and divided into control, glazing, and polishing groups. Surface analysis included roughness, wettability, and surface free energy. Microbiological analysis included CFU (colony-forming units per mL) counts, microbial adhesion at 2, 4, 6, and 8 h, biofilm biovolume, and qualitative biofilm assessment via scanning electron microscopy (sEm). Results: The glazing group showed significantly greater roughness than the polishing (p = 0.006) and control (p = 0.016) groups, along with a lower contact angle (polishing—p = 0.002; control—p < 0.001) and higher surface energy (polishing—p = 0.005; control—p < 0.001). No significant differences were observed in CFU counts for the tested microorganisms (C. albicans, p = 0.158; L. casei, p = 0.610; S. mutans, p = 0.904). Regarding microbial adhesion, the polishing group showed a smaller biofilm-covered area compared to the control group for both total biofilm (p = 0.008) and viable biofilm (p = 0.005). no statistically significant difference was observed in biofilm biovolume (p = 0.082). Conclusions: These findings suggest that, despite the surface differences among the groups, biofilm formation was not significantly affected.