Objective To evaluate the safety and antimicrobial efficacy of low concentration hypochlorous acid (HClO) irrigation for the prevention of open fracture infection in vitro. Methods Mouse epithelioid fibroblast cell line L929 and osteoblast MC3T3-E1 cells were used in this study, and blank control group and HClO groups with different concentrations (10%, 30%, 50% and 100%) were set up. The safety of different concentrations of HClO was evaluated by cell colony formation test, cell proliferation test and apoptosis test in vitro. The red blood cells of mice were taken, and normal saline group, phosphate buffered saline (PBS) group and HClO groups with different concentrations (10%, 30%, 50% and 100%) were set up. The hemolysis risk of different concentrations of HClO was evaluated by hemolysis test. To evaluate the antibacterial effect of low concentration HClO, Staphylococcus aureus and Escherichia coli were cultured in the blank control group, 10% HClO group and antibiotic positive control group to observe the growth of bacteria; and the blank control group, 10% HClO group, antibiotic positive control group, iodophor group and hydrogen peroxide group were set up for the bacteriostatic circle experiment. Results In vitro experiments showed that L929 and MC3T3-E1 cells had colony formation and lower proliferation inhibition and apoptosis rates in the 10% HClO group, while they had no colony formation but higher proliferation inhibition and apoptosis rates in the 30%, 50% and 100% HClO groups (all P<0.01). The erythrocyte hemolysis test was negative at all concentrations in HClO group. Low concentration (10%) HClO could significantly inhibit bacterial growth and had the same antibacterial effect as iodophor and hydrogen peroxide. Conclusion Low concentration (10%) HClO is safe and has good inhibitory effect on both Gram-positive and Gram-negative bacteria. It is a potential irrigation for clinical treatment of open fractures, osteomyelitis and infected bone defects.