Povidone-iodine is a common antiseptic demonstrating success in reducing infection rates in primary arthroplasty; however, recent data suggest that its use in revision arthroplasty may increase infection rates. This study evaluated the effect of povidone-iodine solution on antibiotic cement and investigated the connection between povidone-iodine and increased infection rates in revision arthroplasty. Sixty antibiotic cement samples (ACSs) were formed using gentamicin-impregnated cement. The ACSs were divided into three groups: group A (n=20) was subject to a 3-minute povidone-iodine soak followed by a saline rinse; group B (n=20) underwent a 3-minute saline soak; and group C (n=20) underwent only a saline rinse. The antimicrobial activity of the samples was tested using a Kirby-Bauer-like assay using Staphylococcus epidermidis. The zone of inhibition (ZOI) was measured every 24 hours for 7 days. All groups possessed the greatest antimicrobial activity at 24 hours. Group C displayed a mass-corrected ZOI of 395.2 mm/g, which was statistically greater than the group B ZOI (313.2 mm/g, P<.05) but not the group A ZOI (346.5 mm/g, P>.05). All groups demonstrated a decrease in antimicrobial activity at 48 through 96 hours, with no significant difference at any time point. Prolonged soaking of antibiotic cement in a povidone-iodine or saline solution results in elution of the antibiotic into the irrigation solution, blunting initial antibiotic concentration. When using antibiotic cement, antiseptic soaks or irrigation should be focused prior to cementation. [Orthopedics. 2024;47(1):10-14.].