The purpose of this study was to assess Enterococcus faecalis penetration into the dentin of the apical 3 mm and bacterial death after the application of either chlorhexidine or laser to root-end cavities.Root canals of 60 single-rooted teeth were prepared. In part 1, cementum was removed semicircumferentially from 21 roots, and the smear layer was removed from 15 roots using 17% EDTA/cetrimide. Teeth were inoculated and incubated with E. faecalis for 10 days, rinsed, and live/dead stained. The effect of cementum and smear on bacterial penetration was assessed by confocal laser scanning microscopy (CLSM). In part 2, 39 teeth had root ends resected and cavities ultrasonically prepared. Inoculated roots were assigned to 1 of the following 3 groups: (1) root-end cavities irrigated with 0.2 % chlorhexidine, (2) root-end cavities irradiated with a laser for 20 seconds at 1.5 W, or (3) root-end cavities that received no treatment. Roots were live/dead stained, sectioned, and examined by CLSM. The depth of the bacterial penetration and bacterial survival were compared using the Mann-Whitney U test.The presence of a smear layer and/or cementum did not significantly affect bacterial penetration. In root-end cavities, chlorhexidine was more effective than laser (P < .001), reducing bacterial viability by 93% versus 70% with a laser.E. faecalis invaded the entire width of dentin in the apical 3 mm irrespective of the smear layer and/or cementum. Chlorhexidine was more effective than laser in disinfecting root-end cavities.