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Chronic wounds, defined as those which remain open and inflamed for greater than six weeks, are a major area of clinical concern. Resulting in thousands of amputations per year and billions of dollars spent globally in treatment, chronic wounds are notoriously difficult to successfully treat. Two hallmarks of chronic wounds are that they are thought to harbor biofilm-associated bacteria and tend to be polymicrobial. While the research literature has repeatedly demonstrated the effects of biofilms on wound persistence and the changes to the efficacy of antibiotics, few studies have demonstrated what effect the polymicrobial condition has on the antibiotic tolerance of bacteria. To further explore this, four species of clinically relevant wound pathogens (Pseudomonas aeruginosa, Acinetobacter baumanii, Staphylococcus aureus, and Enterococcus faecalis) were tested in mono- and polymicrobial conditions using the current gold-standard methods for determining antibiotic susceptibility. Noticeable differences in antibiotic tolerance were observed in the polymicrobial condition, including both increased and decreased susceptibility, depending on the antibiotic used. Our data demonstrate that the current clinical methods used for testing antibiotic susceptibility can generate results that are not representative of the infection environment, which may contribute to treatment failure and persistence of polymicrobial infections. [ABSTRACT FROM AUTHOR]