Topical povidone-iodine, chlorhexidine, bacitracin, and vancomycin are commonly used antiseptic and antimicrobial agents to reduce risk and treat surgical site infections in numerous orthopedic procedures. Chondrocytes potentially may be exposed to these agents during operative procedures. The impact of these topical agents on chondrocyte viability is unclear. The goal of this study is to determine human chondrocyte viability ex vivo after exposure to commonly used concentrations of these topical antiseptic and antimicrobial agents. Human osteochondral plugs were harvested from the knee joint of a human decedent within 36 hours of death. Individual human osteochondral plugs were exposed to normal saline as a control; a range of concentrations of povidone-iodine (0.25%, 0.5%, and 1%), chlorhexidine (0.01% and 0.5%), and bacitracin (10,000 units/L, 50,000 units/L, and 100,000 units/L) for 1-minute lavage; or a 48-hour soak in vancomycin (0.16 mg/mL, 0.4 mg/mL, and 1.0 mg/mL) with nutrient media. Chondrocyte viability was evaluated with a live/dead viability assay at 0, 2, 4, and 6 days after exposure to bacitracin at 0, 3, and 6 days). Control subjects showed greater than 70% viability at all time points. Povidone-iodine, 0.5% chlorhexidine, and vancomycin showed significant cytotoxicity, with viability dropping to less than 40% by day 6. Chondrocytes exposed to 0.01% chlorhexidine maintained viability. Chondrocytes exposed to bacitracin showed viability until day 3, when there was a large drop in viability. Commonly used topical concentrations of povidone-iodine, vancomycin, and bacitracin are toxic to human chondrocytes ex vivo. A low concentration of chlorhexidine appears safe. Caution should be used when articular cartilage may be exposed to these agents during surgery. [Orthopedics. 2022;45(5):e263-e268.].