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Abstract For patients who have difficulty in mechanical cleaning of dental appliances, a denture cleaner that can remove biofilm with dense extracellular polymeric substances is needed. The purpose of this study is to evaluate the efficacy of diatom complex with active micro-locomotion for removing biofilms from 3D printed dentures. The diatom complex, which is made by doping MnO₂nanosheets on diatom biosilica, is mixed with H₂O₂to generate fine air bubbles continuously. Denture base resin specimens were 3D printed in a roof shape, andPseudomonas aeruginosa(10⁷ CFU/mL) was cultured on those for biofilm formation. Cleaning solutions of phosphate-buffered saline (negative control, NC), 3% H₂O₂with peracetic acid (positive control, PC), denture cleanser tablet (DCT), 3% H₂O₂with 2 mg/mL diatom complex M (Melosira, DM), 3% H₂O₂with 2 mg/mL diatom complex A (Aulacoseira, DA), and DCT with 2 mg/mL DM were prepared and applied. To assess the efficacy of biofilm removal quantitatively, absorbance after cleaning was measured. To evaluate the stability of long-term use, surface roughness, ΔE, surface micro-hardness, and flexural strength of the 3D printed dentures were measured before and after cleaning. Cytotoxicity was evaluated using Cell Counting Kit-8. All statistical analyses were conducted using SPSS for Windows with one-way ANOVA, followed by Scheffe’s test as a post hoc (p < 0.05). The group treated with 3% H₂O₂with DA demonstrated the lowest absorbance value, followed by the groups treated with 3% H₂O₂with DM, PC, DCT, DCT + DM, and finally NC. As a result of Scheffe’s test to evaluate the significance of difference between the mean values of each group, statistically significant differences were shown in all groups based on the NC group. The DA and DM groups showed the largest mean difference though there was no significant difference between the two groups. Regarding the evaluation of physical and mechanical properties of the denture base resin, no statistically significant differences were observed before and after cleaning. In the cytotoxicity test, the relative cell count was over 70%, reflecting an absence of cytotoxicity. The diatom complex utilizing active micro-locomotion has effective biofilm removal ability and has a minimal effect in physical and mechanical properties of the substrate with no cytotoxicity.