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Abstract Background In dentistry, nosocomial infection poses a great challenge to clinicians. The microbial contamination of water in dental unit waterlines (DUWLs) is ubiquitous. Such infected DUWLs can transmit oral microbes in the form of aerosols. Previous studies have suggested treating DUWLs with various disinfectants to reduce cross-contamination. The literature lacks a comparative evaluation of the effect of the use of 0.2% chlorhexidine (CHX) and 0.1% sodium hypochlorite (NaOCl) in DUWLs on aerosolized bacteria generated during dental procedures. Objective To compare the effect of NaOCl and CHX in DUWLs on aerosolized bacteria generated during restorative and endodontic procedures. Materials and methods A total of 132 patients were equally divided into three groups (n = 44 in each group) according to the content of DUWL as follows. Group I—0.1% NaOCl Group II—0.2% CHX Group III—distilled water (Positive control) One-way ANOVA was performed and the Kruskal–Wallis test was used for intergroup comparison. Results For the restorative procedure, inter-group comparison of mean colony-forming units (CFU) scores showed a statistically significant difference between the groups (p - .001) with the score of group 3 higher than group 2 followed by group 1. For the endodontics, an inter-group comparison of CFU scores showed a statistically significant difference between the groups (p - .003) with the mean score in group 1 being the lowest and group 3 being the highest. Conclusion The addition of NaOCl or CHX in DUWLs shows an effective reduction in aerosolized bacteria compared to distilled water.