Ultrasound technologies have a wide range of hospital and dental applications which include cleaning and disinfection of surgical and dental instruments. We measured the germicidal efficacy of sonication, with or without chemical disinfectants, in an ultrasonic bath delivering a frequency of 35 kHz and an intensity of 0.66 W/cm2. Cultures of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans were exposed to ultrasound and to an amine-based disinfectant in non-bactericidal concentrations. Ultrasonication for 60 min alone did not cause a significant killing of the bacteria and yeast. However, we were able to show that sonication can act as a powerful synergistic agent to increase the cidal efficacy of the disinfectant against S. aureus and P. aeruginosa. C. albicans was more resistant to the combination of ultrasound and chemical disinfection. The key role in the action of ultrasound in cleaning of instruments and perhaps in enhanced disinfection is played by cavitation phenomena. The distribution of cavitations in an ultrasonic bath is not homogenous. We found a similar synergistic effect of ultrasound and disinfectant in positions with low cavitation. The synergistic effect was not reduced inside rubber tubes. Before ultrasound can be accepted as an integral part of the cleaning and disinfection process of medical instruments, the influence of intensity and frequency of sonication and the effects of cavitation must be clarified.