The aim of this study was to assess, by scanning electron microscopy analysis, the cleaning ability of irrigants and auxiliary chemical substances in biomechanical preparation. Thirty-two single-rooted human teeth were subjected to standardized root canal instrumentation with different irrigants (n = 8): group 1, 2.5% NaOCl + 17% ethylenediaminetetraacetic acid (EDTA); group 2, 2% chlorhexidine gel + 17% EDTA; group 3, Canal Plus + 2.5% NaOCl; group 4, saline + 17% EDTA. After instrumentation, the teeth were prepared for scanning electron microscopy analysis (2000x) to evaluate the cleaning of cervical, middle, and apical thirds. The area analyzed was quantified according to the percentage of open tubules, and data were statistically analyzed by analysis of variance and Tukey tests (P = .05). The results showed statistically significant difference with higher quantity of open tubules in groups 1 and 3. In all groups, the cleaning obtained on the cervical third was better than that obtained on the middle and apical thirds, with statistically significant difference in the chlorhexidine group. Irrigation with Canal Plus in combination with NaOCl and NaOCl followed by EDTA produced greater cleaning of the root canal walls.