Aim: The aim of this study was to evaluate the hydrogen peroxide and cetylpyridinium chloride as bacterial decontaminants of dental unit water lines at a private Peruvian dental school. Materials and Methods: Water samples were obtained from 66 dental units of a University Dental Clinic before decontamination treatment and at days 3 and 7 thereafter. The biofilm treatments were applied equitably among the two treatment groups (n = 22) and one negative control (distilled water). The samples obtained on each collection day were taken to the biochemical laboratory in thermal boxes and then diluted, seeded, and incubated at 37°C for 24 h to count colony forming units per milliliter (CFU/ mL). Results: The samples to which hydrogen peroxide were applied had a mean of 1.53 × 105 CFU/mL before application, 0.04 × 105 CFU/mL at day 3, and 0.03 × 105 CFU/mL at day 7, whereas the samples undergoing cetylpyridinium chloride treatment had a mean of 1.74 × 105 CFU/mL before application, 615.38 CFU/mL on day 3, and 307.69 CFU/mL on day 7. Distilled water treatment showed a mean of 1.72 × 105 ± 0.39 × 105 CFU/mL at baseline, 1.51 × 105 ± 1.40 × 105 CFU/mL at day 3, and a mean of 1.74 × 105 ± 0.47 × 105 CFU/mL at day 7. Statistically significant differences were found among the three treatment groups at days 3 (P ≤ 0.001) and 7 (P ≤ 0.001) but not at baseline (P = 0.306). Conclusions: The antibacterial effect of cetylpyridinium chloride was significantly greater than that of hydrogen peroxide and distilled water, and can, therefore, be used for bacterial control in the water lines of dental units.