Objective: Biofilm formation in dental waterlines brings opportunistic infections, especially for immunosuppressive patients. This study aimed to determine biofilm-forming microorganisms by various methods and investigate disinfectants' effects on biofilm. Materials & Methods: In the study, samples were obtained from the waterlines of 10–15 aged six dental units, before (0 min.) and after chlorine dioxide (ClO2) and hypochlorous acid (HOCl) treatment (1, 5, 10, 20, and 30 min.), and total colony counts were performed using conventional surface smear method (SSM) and dip slide method (DSM). The Congo red agar and Christensen methods were used to examine the biofilm-forming properties of the isolates. Monitoring of biofilm presence was also visualized by SEM scanning. Results: When DSM and SSM are compared in all units where ClO2 and HOCl are applied, DSM can detect bacterial growth even during periods of greater exposure to disinfectant application. Although DSM can achieve a value approaching 3% even at the 10th minute in units treated with HOCl; SSM does not show reproduction at the same disinfectant exposure and duration; It was observed that in the units where ClO2 was applied, the growth was no longer observed at the 10th minute with DSM, and SSM, 50% growth in the first minute of the units treated with ClO2 could not be detected in the 5th minute. Conclusions: It is concluded that it can be advisable to routinely disinfect the dental unit water systems with non-toxic doses of ClO2 application before patient treatments in clinics and also to perform contamination controls at regular intervals with DSM, which is a sensitive and very practical method. Research highlights: It has been observed that the dip slide method can count bacteria more sensitively than conventional methods in dental water systems without the need for experienced personnel and equipment. The difference between biofilm formation in water systems before and after disinfectant exposure in SEM examinations is remarkable. The effects of ClO2 and HOCl on biofilm were investigated and bacterial growth was inhibited in dental units between 5 and 10 minutes with both disinfectants.