This study assessed the effectiveness of different concentrations of sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) (Vista Dental Products, Racine, WI), and BioPure MTAD (Dentsply Endodontics-Tulsa Dental, Tulsa, OK). Intracanal contents were collected from 10 patients diagnosed with chronic apical periodontitis. The samples were cultured on hemisections of root apices to generate a polymicrobial biofilm. Each biofilm was separately immersed in 6% NaOCl, 3% NaOCl, 1% NaOCl, 2% CHX, 1% NaOCl followed by BioPure MTAD, and sterile phosphate buffered solution (PBS). SEM analysis showed 6% NaOCl and 3% NaOCl were capable of disrupting and removing the biofilm; 1% NaOCl and 1% NaOCl followed by MTAD were capable of disrupting the biofilm, but not eliminating bacteria; 2% CHX was not capable of disrupting the biofilm. Viable bacteria could not be cultured from specimens exposed to 6% NaOCl, 2 % CHX, or 1% NaOCl followed by BioPure MTAD. These results indicate that 6% NaOCl was the only irrigant capable of both rendering bacteria nonviable and physically removing the biofilm.