Denture stomatitis is a common manifestation of oral candidiasis affecting some 65% of denture wearers. This condition is initiated by the adherence of Candida albicans to denture base acrylic resin. The present study aimed to test the in vitro effect of traditional and novel fabrication methods on Candida albicans adhesion to denture base samples. Denture based acrylic discs were fabricated using: (i) computerized milling, (ii) 3D printing, (iii) heat curing, and (iv) cold curing. Discs were tested for surface roughness (Ra), hydrophobicity (contact angle), mucin adsorption (Bradford assay), and Candida albicans adhesion. 3D printing significantly increased microbial cell adhesion as compared with heat curing, and computerized milling significantly decreased it. These results were associated with mucin adsorption levels rather than surface roughness. Results suggest that 3D printing may increase the risk for developing denture stomatitis, whereas computerized milling may decrease it as compared with traditional heat curing denture base fabrication.