Abstract The purpose of this prospective, randomized, single-blind study was to compare the in vivo antibacterial efficacy of a hand/rotary technique versus a hand/rotary/ultrasound technique in mesial root canals of necrotic mandibular molars. The hand/rotary group consisted of 16 mesial roots prepared with a hand/rotary technique. The hand/rotary/ultrasound group consisted of 15 mesial roots prepared similarly, followed by 1 minute of ultrasonic irrigation per canal with an ultrasonic needle in a MiniEndo unit and 15 mL/canal of 6.0% sodium hypochlorite. Canals were sampled before and after instrumentation and after 1 minute of ultrasonic irrigation. Samples were incubated anaerobically on reduced blood agar for 7 days at 37°C, and colony-forming units (CFUs) were counted. The addition of 1 minute of ultrasonic irrigation resulted in significant (p = .0006) reduction in CFU count and positive cultures (p = .0047). Logistic regression analysis indicated the addition of ultrasonic irrigation was 7 times more likely to yield a negative culture.