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Objective: This study evaluated the antimicrobial effect and cytotoxicity of hypochlorous acid(HClO) obtained from an innovative electrolytic device., Design: The root canals of fifty extracted human teeth were inoculated with Enterococcus faecalis and divided into 5 groups (n = 10): DW (control); 2% chlorhexidine gel(CHX); 2.5% sodium hypochlorite(NaOCl); 250 ppm HClO and 500 ppm HClO. The counting of colony forming units evaluated the decontamination potential of each group. Cytotoxicity was evaluated after inoculation of tested protocols in fibroblastic cells for 3 min, calculating the cell viability. Specific statistical analysis was performed (α = 5%)., Results: The highest bacterial reduction was observed in experimental groups, with no statistical differences from each other (p > 0.05). The highest number of viable cells was observed in control group, followed by 250 ppm HClO and 500 ppm HClO groups, with statistical differences from each other (p < 0.05)., Conclusions: It could be concluded that HClO presented high antimicrobial activity and low cytotoxicity at both tested concentrations., Competing Interests: Declaration of Competing Interest Dr. Liviu Steier owns IP rights on the Dentaqua device. The rest of authors state that they have no financial affiliation (e.g., employment, direct payment, stock holdings, retainers, consultantships, patent licensing arrangements or honoraria) or involvement with any commercial organization with direct financial interest in the subject or materials discussed in this manuscript, nor have any such arrangements existed in the past three years., (Copyright © 2024 Elsevier Ltd. All rights reserved.)