Bacterial contamination is a problem in dental unit water lines with the consequence of implementing regular disinfection. In this study, the short-term impact of chlorine dioxide (ClO₂) treatment was investigated on the microorganisms Legionella pneumophila and L. anisa, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. The environmental background was proven as an important factor regarding the tolerance to 0.4 mg/L ClO₂ as saline and phosphate-buffered saline resulted in a higher bacterial reduction than tap water. Gram-positive microorganisms demonstrated higher robustness to ClO₂ than Gram-negative, and microorganisms adapted to tap water showed increased stability compared to cultured cells. At high densities, substantial numbers of bacteria were able to withstand disinfection, whereby the use of 4.6 mg/L ClO₂ increased the inactivation rate. A massive cell decrease occurred within the first 5 minutes with subsequent plateau formation or slowed cell reduction upon further exposure. This biphasic kinetics cannot be explained by a ClO₂ depletion effect alone, because the probability of bacterial subpopulations with increased tolerance should be taken into account, too. Our results prove high disinfection efficiency to microorganisms that were rather found in correlation to the level of bacterial contamination and background solutions than the chosen concentration for ClO₂ treatment itself.