Beyond mechanical performance and aesthetics, the susceptibility of 3D-printed resins to microbial colonization and biofilm formation represent an important factor influencing dentures’ longevity. Therefore, this study evaluated Candida albicans colonization and mature biofilm formation on three different 3D-printed denture base resins (Bio Denture—BD; Denture Base Cosmos—CD; Smart Print Bio Denture—SP) and compared them to heat-curing resin (HC). Before the microbiological evaluation, the surface roughness (Sa) was assessed. Biofilm viability was determined through colony-forming units per milliliter (CFU/mL) and biofilm morphology was qualitatively examined using a scanning electron microscope (SEM). The composition of the extracellular polymeric substance (EPS) was investigated by measuring the amounts of carbohydrates (µg/mL), proteins (ng/mL), and extracellular DNA (eDNA) (fluorescence unit). One-way ANOVA was performed for eDNA and Sa and Kruskal–Wallis for the other properties (α = 0.05). Higher surface roughness mean values (standard deviation) (p < 0.05) were observed in CD [0.111 (0.013)] compared to HC [0.084 (0.018) and BD [0.078 (0.015)]. For wettability, BD [63.2 (5.2)] and SP [65.2 (3.1)] resins showed a greater wettability (p < 0.05) than HC resin [73.0 (3.5)], while SP showed lower (p < 0.01) protein levels (425 ng/mL) compared to HC (568.6 ng/mL) and BD (554.8 ng/mL) in the EPS. Despite these differences, the 3D-printed denture base resins exhibited microbial load (CFU/mL), EPS composition (carbohydrates and eDNA), and morphological features of C. albicans biofilm comparable to those of conventional heat-cured PMMA. These findings suggest that, despite resin-specific variations, 3D-printed denture base materials exhibit a similar susceptibility to C. albicans colonization and biofilm formation as conventional denture bases, thereby directing future research towards developing new 3D-printed resins with enhanced antimicrobial properties to improve clinical outcomes.