Background: Denture base resin has some drawbacks. This study investigated the impact of nanodiamonds (ND) and autoclave polymerization on the surface characteristics, translucency, and Candida albicans adherence in polymethyl methacrylate (PMMA) denture base resin after thermocycling. Methods: Heat-polymerized PMMA discs (15 × 2 mm) with a total sample size n = 160 were studied. Specimens were categorized into two main groups (N = 80): conventional water-bath-polymerized PMMA (CP/PMMA) and autoclave-polymerized PMMA (AP/PMMA). Each group was subdivided according to the ND concentration into four groups (n = 20): unmodified PMMA as a control, and 0.1%, 0.25%, and 0.5% ND–PMMA. Scanning electron microscopy (SEM) was used to inspect the morphology of the ND and the ND–PMMA mixtures before heat polymerization. The specimens were exposed to thermal cycling (5000 cycles at 5 and 55 °C), then surface roughness was measured with a non-contact optical interferometric profilometer, contact angle with an automated goniometer, and translucency using a spectrophotometer. Colony-forming units (CFU) were used to determine the adherence of Candida albicans cells to the specimens. ANOVA and Tukey post hoc tests for pairwise comparison were utilized for the statistical analysis (α = 0.05). Results: Surface roughness was significantly reduced with ND addition to CP/PMMA (p ˂ 0.001), while the reduction was not statistically significant in AP/PMMA (p = 0.831). The addition of ND significantly reduced the contact angle, translucency, and Candida albicans count of CP/PMMA and AP/PMMA (p ˂ 0.001). The incorporation of ND in conjunction with autoclave polymerization of PMMA showed significant reduction in all tested properties (surface roughness, contact angle and Candida albicans adherence) except translucency (p = 0.726). Conclusions: ND addition to PMMA and autoclave polymerization improved the surface properties with respect to antifungal activities, while the translucency was adversely affected.