The utilization of three-dimensional (3D) printing in dentistry for denture base fabrication is rapidly gaining traction in recent years. To date, a wide range of 3D printing technologies and materials can be utilized for the fabrication of denture bases. However, there is a lack of understanding of the effect of printability, mechanical, physical, and biological properties of the 3D-printed denture base upon fabricating with different vat polymerization techniques. This study was carried out to elucidate the effect of different vat polymerization techniques in fabricating denture bases. In this study, the NextDent denture base resin was printed with stereolithography (SLA), digital light processing (DLP), and light-crystal display (LCD) techniques and underwent the same post-processing procedure. One-way ANOVA and Tukey’s post hoc were used to analyze the data statistically. The results showed that the greatest flexural strength was exhibited by the SLA (150.8 ± 7.93 MPa) , followed by the DLP and LCD. This trend was consistent in fracture toughness and microhardness, with SLA demonstrating superior strength, followed by DLP and LCD. Notably, the water sorption and solubility of the DLP are significantly higher (p < 0.05) than other groups (31.51 ± 0.92 μg mm3) and 􁉀5.32 ± 0.61 μg mm3􁉁, respectively. SLA demonstrated the highest overall mechanical strength among all tested groups, albeit at a slower printing rate, owing to its ability to achieve a higher degree of conversion. Surface morphology analysis revealed no discernible differences after undergoing thorough mechanical polishing, indicating that the layered structure was confined to the outer surface. However, the most Candida albicans adhesion was also found in SLA (221.94 ± 65.80 CFU/ml) probably due to its higher surface roughness along the successive layers. Nonetheless, all denture bases fabricated with different vat polymerization did not demonstrate any cytotoxic effect on the Human Gingiva Fibroblast. In conclusion, this study confirmed that the NextDent denture base resin designed for DLP can be printed with different vat polymerization techniques and all tested groups met the ISO requirement aside from the water solubility. SLA exhibited the greatest mechanical strength while the DLP showed the lowest microbial adhesion.