Objective: This in vitro study evaluated the effect of different endodontic auxiliary chemical substances over Enterococcus faecalis (Ef) biofilm through confocal laser scanning microscopy (CLSM). Methods: Forty‐five bovine incisors were infected with Ef for 21 days. Teeth were divided into five groups: group 1: 2.5% NaOCl + EDTA, group 2: 2% CHX gel + EDTA, group 3: 2% CHX liquid + EDTA, group 4: 2.5% NaOCl + 2% CHX gel + EDTA, group 5: 2.5% NaOCl + 2% CHX liquid + EDTA and a negative and a positive control group (NCG; PCG). The samples were stained with SYTO9 and propidium iodide and analyzed by CLSM. Bacterial viability was quantitatively analyzed by the proportions of dead and live bacteria in the biofilm remnants. Scores were standardized according to the total bacterial load (TBL)—1: ≤25%, 2: >25 ≤50%, 3: >50 ≤75%, 4: >75% and debris—1: absence of debris; 2: presence of debris. Statistical analysis was carried out through the Kruskal–Wallis and the Fischer exact tests (P = 0.05). Results: No statistical differences were observed to CFU, debris and bacterial viability. Conclusion: None of the tested substances could completely eliminate Ef from the root canal space. Microsc. Res. Tech. 76:658–662, 2013. © 2013 Wiley Periodicals, Inc.