In dentistry, interest in computer-aided design and computer-aided manufacturing (CAD/CAM) over traditional denture fabrication systems is increasing. However, few studies have compared the physiochemical and biological properties of different fabrication methods in regards to Candida albicans. C. albicans is the most common oral fungal pathogen, often related to dentures and elderly patients. Although various factors have been reported to influence C. albicans proliferation, conclusive evidence is lacking. Therefore, the aim of this study was to evaluate and compare the physicochemical properties of different denture materials in terms of surface roughness and C. albicans adhesion on the basis of polymerization methods and 3D print orientation. Four types of resin disks were prepared: autopolymerization, heat-activated polymerization, milling, and 3D printing. The surface roughness and water contact angle were measured via a confocal laser scanning microscopy (CLSM) and the sessile drop method. After C. albicans inoculation, microbial adhesion was measured by scanning electron microscope (SEM), a crystal violet assay, and an alcian blue assay. To further investigate the relationship between surface roughness and microbial adhesion, 3D-printed resin was fabricated at different build orientations. The resins were printed at 0, 45, and 90-degrees to modulate different surface roughness and simulate commonly used clinical settings. The same experimental set was used. The cell wall thickness of each group was measured via CLSM. For statistical analysis, one-way ANOVA and Tukey’s post hoc tests were performed. The 3D-printed group presented the greatest adhesion with the highest roughness parameters (Ra, Rdq). The milled group presented the lowest adhesion with the lowest surface roughness values. (p < 0.05) Among the 3D-printed samples with different build orientations, the 0-degree presented the lowest surface roughness and the lowest microbial adhesion. (p < 0.05) Microbial adhesion was less related to average roughness and more related to microroughness. The findings were further confirmed with SEM images with prominent grooves and layer lines with speckled patterns on 45 and 90-degree. The cell wall thickness showed no difference between the groups. (p > 0.05). Microbial adhesion is significantly affected by fabrication methods and build orientation. Milled and 0-degree 3D printed resins provide improved options with the potential to minimize Candida albicans adhesion with the lowest surface roughness. Due to their high surface roughness and microbial adhesion, 3D-printed resins with different printing orientations require meticulous polishing and regular cleansing. Despite the limitation of in vitro study, this study aims to provide a deeper understanding of surface morphology and microbial adhesion and provide clinical insights that milled and 0-degree printed resin are more favorable in minimizing C. albicans adhesion.