Pseudomonas aeruginosa is a pathogen notorious for its antimicrobial resistance and is currently classified as a high-priority pathogen for which new drugs are needed. Tetrasodium EDTA (tEDTA) is one of the new antimicrobial compounds that have been shown to have good antibacterial and antibiofilm efficacy against P. aeruginosa. Due to the diversity and highly drug-tolerant nature of P. aeruginosa biofilms in different infection environments, it is important to carry out pre-clinical testing of new antibiofilm agents against this pathogen in media and models that accurately mimic diverse infection environments. In this study, we used different high validity media and biofilm models that mimic chronic wounds, endotracheal tubes, and cystic fibrosis lung infections to assess the efficacy of tEDTA against P. aeruginosa biofilms. We report that different infection environments influence the susceptibility of both planktonic and biofilm forms of P. aeruginosa to tEDTA. The highest tolerance to tEDTA was observed in the media and biofilm model that mimics the endotracheal tube environment. In conclusion, we show that although different infection environments influence the efficacy of tEDTA against P. aeruginosa biofilms, it has good potential for use as an alternative antimicrobial in treating P. aeruginosa-associated biofilm infections. ### Competing Interest Statement The authors have declared no competing interest.

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