Ninety-four single-canal roots were prepared using the step-down technique. Forty-two canals were irrigated with 2% chlorhexidine, 42 canals with 5.25% sodium hypochlorite (NaOCl), and 10 control canals with phosphate-buffered saline (PBS). The chlorhexidine and NaOCl groups were each then equally divided into a final irrigation group and a 1-min passive ultrasonic irrigation group. Canals were enlarged with a Parapost drill. The apical 3-5 mm was covered with nail polish. Canals were rinsed with PBS, dried, refilled with PBS, and stored. At 6 h, 20 μl of fluid was pipetted from each canal and placed into wells on agar plates, which were inoculated with Streptococcus sanguinis. The plates were incubated, and zones of inhibition were measured. Sampling was repeated at 24, 48, 72, 96, 120, 144, and 168 h. Residual antimicrobial activity with 2% chlorhexidine was statistically significantly superior to 5.25% NaOCl with irrigation alone and with final passive ultrasonic activation (p < 0.001). Chlorhexidine experimental groups demonstrated residual antimicrobial activity for as long as 168 h.