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If a tissue biopsy from a chronic wound is sampled for culture, the antibacterial properties of local anesthetics may pose a problem in producing false-negative results. The purpose of this study was to investigate the effects of EMLA® (AstraZeneca) and lidocaine on common wound pathogenic bacteria. An in vitro study of a total of 25 clinical isolates and ATCC reference strains of Staphylococcus aureus (including methicillin-resistant S. aureus), Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pyogenes was conducted. The isolates were exposed to the local anesthetic drugs (and some of their contents separately) at 35°C over a 24-hour period and time–kill curves were recorded. No culture media were used and saline was used for controls. EMLA® was found to have a rapid acting and powerful antibacterial effect and should not be used before culturing tissue samples. Lidocaine 1% was found not to inhibit the bacterial strains when exposure time was held below 2 hours. We conclude that culturing tissue from a wound biopsy is safe within 2 hours when a pure, preservative-free lidocaine 1% solution is used.