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Aims. In wound irrigation, 1 mM ethylenediaminetetraacetic acid (EDTA) is more efficacious than normal saline (NS) in removing bacteria from a contaminated wound. However, the optimal EDTA concentration remains unknown for different animal wound models. Methods. The cell toxicity of different concentrations of EDTA dissolved in NS (EDTA-NS) was assessed by Cell Counting Kit-8 (CCK-8). Various concentrations of EDTA-NS irrigation solution were compared in three female Sprague-Dawley rat models: 1) a skin defect; 2) a bone exposed; and 3) a wound with an intra-articular implant. All three models were contaminated with Staphylococcus aureus or Escherichia coli. EDTA was dissolved at a concentration of 0 (as control), 0.1, 0.5, 1, 2, 5, 10, 50, and 100 mM in sterile NS. Samples were collected from the wounds and cultured. The bacterial culture-positive rate (colony formation) and infection rate (pus formation) of each treatment group were compared after irrigation and debridement. Results. Cell viability intervened below 10 mM concentrations of EDTA-NS showed no cytotoxicity. Concentrations of 1, 2, and 5 mM EDTA-NS had lower rates of infection and positive cultures for S. aureus and E. coli compared with other concentrations in the skin defect model. For the bone exposed model, 0.5, 1, and 2 mM EDTA-NS had lower rates of infection and positive cultures. For intra-articular implant models 10 and 50 mM, EDTA-NS had the lowest rates of infection and positive cultures. Conclusion. The concentrations of EDTA-NS below 10 mM are safe for irrigation. The optimal concentration of EDTA-NS varies by type of wound after experimental inoculation of three types of wound. Cite this article: Bone Joint Res 2021;10(1):68–76