Objectives: Since ultrasonic dental scalers contain internal narrow-diameter waterline tubing susceptible to bacterial biofilm growth, this study determined the cultivable prevalence of aerobic mesophilic heterotrophic bacteria in ultrasonic scaler output water subjected to one of two commercial biocides favorably reviewed by the American Dental Association as effective for microbial biofilm control in dental unit waterlines, and assessed the potential antimicrobial effect on output water of an ultrasonic scaler tip activated at a maximum power frequency. Methods: Aseptically-collected water samples were obtained, and neutralized for residual chlorine with sodium thiosulfate, from 12 ultrasonic scalers provided municipal tap water treated with an iodine resin cartridge (Group A), and from eight ultrasonic scalers provided distilled water treated with a 0.78% silver ion-based disinfectant (Group B), all within a dental school clinic setting. The latter eight ultrasonic scalers were additionally sampled after output water was passed through an inserted scaler tip activated to a maximum power setting similar to its potential use in clinical patient care. All water samples were evaluated for aerobic mesophilic heterotrophic bacteria using nationally-accepted analytical procedures detailed in Standard Methods for the Examination of Water and Wastewater, 23rd Edition (2017), with sample aliquots spread-plated onto R2A agar and aerobically incubated at 28°C for seven days. Aerobic mesophilic heterotrophic bacteria were considered elevated when ≥ 500 CFU/ml of the species were recovered per water sample. Predominant cultivable isolates of aerobic mesophilic heterotrophic bacteria in the water samples were identified using matrix- assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry and Bruker MALDI Biotyper analytic software. Results: Aerobic mesophilic heterotrophic bacteria averaged 610 ± 380 (SD) CFU/ml per ultrasonic scaler in Group A ultrasonic scalers, with eight of 12 (66.7%) evaluated ultrasonic scalers yielding elevated counts ≥ 500 CFU/ml. Similar mean values of 667 ± 340 (SD) CFU/ml per ultrasonic scaler were found in Group B ultrasonic scalers, with five of eight (62.5%) evaluated ultrasonic scalers yielding elevated aerobic mesophilic heterotrophic bacteria counts ≥ 500 CFU/ml. Acidovorax temerans, Novosphingobium subterraneum, Cupriavidus metallidurans, and Sphingobium yanoikuyae were the most prevalent cultivable species identified from ultrasonic scaler waterlines where the source water was municipal tap water treated with molecular iodine. In contrast, nearly pure growth of Cupriavidus metallidurans alone dominated cultivable waterline isolates from ultrasonic scalers where the source water was distilled water treated with a 0.78% silver ion disinfectant. No statistically significant impact was found on aerobic mesophilic heterotrophic bacterial counts after ultrasonic scaler output water passed through an inserted scaler tip activated to maximum power (P > 0.05, Wilcoxcon signed-rank test). Conclusions: Approximately two-thirds of ultrasonic scalers in a dental school clinic setting yielded output water that failed to meet potable water regulatory standards in the United States with regard to aerobic mesophilic heterotrophic bacterial counts, even when subjected to disinfection with one of two American Dental Association favorably-reviewed biocide products, or exposed to high-frequency ultrasonic scaler tip oscillations. These findings question the effectiveness of infection control procedures used to manage ultrasonic scaler waterlines, and raise concerns about the safety of ultrasonic scaler output water introduced into the oral cavity of dental patients during dental treatment procedures. / Oral Biology