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Background: Povidone-iodide (Betadine) is an antiseptic that is applied topically and has many uses in the medical community, such as in wound care and pre- and post-operative surgical procedures. This study was done to measure the effectiveness of Betadine solutions in inhibiting the growth of Gram-negative and Gram-positive bacteria. Methods: The ability of 2.5 and 10% Betadine solutions to inhibit bacterial growth was measured against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Acinetobacter baumannii. We grew the bacteria independently and together to simulate a hospital environment. Results: All the bacteria showed zones of inhibition. However, discs were also tested for live bacteria using the colony-forming unit assay. Complete killing was only seen for S. aureus with the 10% Betadine solution. All other bacteria showed growth on the disc. Conclusions: This study showed several things. First, the zone of inhibition assay does not give an accurate assessment of antimicrobial properties when used alone and should be followed by a colony-forming unit assay. Second, 2.5% and 5% Betadine do not have effective antimicrobial properties against any of the bacteria tested, and 10% Betadine is only effective against S. aureus and not effective against the other bacteria tested.