ObjectiveThis research endeavors to scrutinize the influence of attachment systems and denture cleaning methodologies on microbial biomass and composition within the realm of implant-supported overdentures, a crucial consideration for patients with dentition defects necessitating such prosthetic solutions.Subjects and methodsEmploying five polymethyl methacrylate specimens designed to emulate the fitting surfaces of traditional dentures and implant-supported overdentures. Following the polishing of each specimen and the quantification of its roughness, co-cultivation with three distinct microbial strains ensued, culminating in ultrasonic cleaning in water. The bar-clip group, differentiated by the depth of attachment, underwent cleaning employing four diverse methods. Biomass quantities were meticulously recorded both pre and post cleaning interventions, with subsequent data analysis via t-testing and one-way ANOVA, maintaining a significance level of α = 0.05.ResultsThe bar-clip groups demonstrated an elevated degree of microbial adhesion, with the deeper locator group exhibiting heightened biomass residue post-cleaning, indicative of increased cleaning complexity. Ultrasonic cleaning predominantly targeted biofilm and deceased bacteria, whereas chemical cleaners primarily reduced the quantity of viable bacteria. The synergistic application of ultrasonics and chemical cleaning treatments yielded the minimal biomass residue.ConclusionIn contemplating the utilization of dentures milled by dental computer-aided design/manufacturing systems, meticulous pre-use surface polishing is imperative. The extent of biofilm adhesion correlates with the chosen attachment system. This study advocates for the incorporation of ultrasonic cleaning in conjunction with chemical cleaning solutions to optimize the removal of biofilm and live cellular entities in the context of implant-supported overdentures.