Background: Sodium hypochlorite (NaOCl) is the most commonly used irrigant in endodontics. The purpose of this study was to evaluate the effect of NaOCl solution (2.5%) and gel (3%) with/without passive ultrasonic irrigation (PUI) on Enterococcus faecalis, Escherichia coli, and their endotoxins, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Methods: 40 human lower premolars were contaminated with E. coli (ATCC 25922) for 28 days and E. faecalis (ATCC 29212) for 21 days. Specimens were randomly divided into four groups: (1) 2.5% NaOCl irrigating the canals without PUI activation; (2) 2.5% NaOCl with PUI; (3) 3% NaOCl gel irrigating the canals without PUI; and (4) 3% NaOCl gel with PUI. 40 mL of irrigant was used for each group. PUI activation was carried out using E1-Irrisonic stainless-steel tip at 10% frequency. After treatment, all specimens were filled with 3mL of 17% ethylenediaminetetraacetic acid (EDTA) for 3min and then washed with nonpyrogenic saline solution. Three samples were collected from the canals: S1, at baseline to confirm biofilm formation; S2 after treatment; and S3 after EDTA. Samples were assessed for E. coli and E. faecalis colony forming units, and LPS and LTA were assessed using chromogenic kinetic LAL assay and ELISA, respectively. Data were analyzed by Kruskal-Wallis, Friedmann and Dunn tests with α≤0.05. Results: All groups were effective in reducing the microbial load of E. coli and E. faecalis after treatment without a significant difference among the groups. NaOCl and NaOCl gel groups had no significant difference in reducing LPS and LTA. Statistically increased reduction was seen for NaOCL + PUI and NaOCl gel + PUI compared for groups without PUI. Conclusions: NaOCl gel has the same antimicrobial action of NaOCl solution and can partially detoxify endotoxins. PUI improves NaOCl (gel or solution) action over E. faecalis and E. coli and their endotoxins.