Background: The roughness of denture base materials is mainly affected by inherent material features, the polishing technique, and the operator’s manual skills. The surface irregularities of the denture base resin’s roughness profile is one of the components characterizing the superficial topography of dental prostheses, and it is a critical parameter for describing surface morphology. Generally, the increment of the surface roughness minimizes cleanability and promotes the rate of bacterial adherence, affecting the expression of bacterial adhesins. The purpose of this in vitro study was to investigate the roughness of four commercially available denture base resins employed for removable prosthodontics. Methods: Twenty-five specimens were realized and submitted to observation for three dimensions (vertically, horizontally, 45°). Average surface roughness (Ra), Rv, Rp, and Rq were measured with a calibrated mechanical roughness tester (Taylor Hobson Surtronic 25; Taylor Hobson, Leicester, UK). Data were analyzed through a Kruskal–Wallis test. Results: Significant differences in Ra between the groups were assessed. Baltic Denture System™ (Merz Dental GmbH; Lütjenburg, Germania) showed a lower surface roughness coefficient and a more homogeneous surface. Conclusions: The functional importance of surface roughness parameters for denture base materials must be explored, not only considering recognized surface features.