Objectives/Aims: This in vitro laboratory study compared the efficacy of water, sodium percarbonate (SPC) and chlorine dioxide (ClO₂) solutions in the disinfection of dental unit water lines (DUWLs). Materials and Methods: New DUWL tubes were cut, split open, and mono-culture and mixed-culture biofilms of Staphylococcus aureus, Enterococcus faecalis and Streptococcus mutans were grown. Harvested biofilms from the sectioned DUWL tubes were exposed to sterile distilled water, SPC or 5 and 10 p.p.m. ClO₂ in both a stationary phase and through a constant flow. Bacterial counts were compared using the Kruskal–Wallis nonparametric rank test. Results: In the mono-culture biofilms, SPC, 5 and 10 p.p.m. ClO₂ significantly reduced all the test organisms (P<0.01). However, no significant difference was found between SPC and ClO₂. In the mixed-culture biofilms exposed to disinfectant without flow, ClO₂ significantly reduced the biofilm (P=0.02) compared with water and SPC. Similarly, in the constant flow study, ClO₂ proved to be superior to water. Conclusion: At low concentrations, ClO₂ with and without flow significantly reduced the mixed-culture biofilm grown in vitro on the sections of the DUWL tubes. Therefore, it has the potential to be used in the patient treatment water, as it is potable at these concentrations, and to decontaminate and limit the biofilm formation in the water lines.