In-office dental unit waterline (DUWL) testing systems are commercially available for monitoring DUWL bacteria. The current study compared Aquasafe, Petrifilm, and Heterotrophic Plate Count Sampler (HPCS) with R2A plating methodology, considered the gold standard for enumerating heterotrophic bacteria in potable water. Samples were collected from 20 dental units. Heterotrophic bacterial counts of ≤500 CFUs/mL were used as the cut-off for assessing in-office testing compared to R2A laboratory plating. Validity was assessed using sensitivity and specificity, along with positive and negative predictive values. Results were also compared using concordance and kappa statistics. All in-office tests demonstrated 100% specificity and positive predictive values, while sensitivity and negative predictive values were low (Petrifilm, 57%/50%; HPCS, 50%/46%; Aquasafe, 21%/35%). Concordance and kappa values for agreement with R2A plating were as follows: Petrifilm 70% (κ = 0.44), HPCS 65% (κ = 0.38), and Aquasafe 45% (κ = 0.14). In-office DUWL testing with Aquasafe, Petrifilm, and HPCS agreed poorly with R2A plating methodology and is not valid or reliable as a means of accurately monitoring bacterial density in DUWL. These in-office test systems should not be used for assessing compliance with the ADA and CDC standard for acceptable heterotrophic bacterial counts in DUWLs (≤500 CFUs/mL).