Background: Povidone-iodine (PI), chlorhexidine gluconate (CHG), and vancomycin (VANC) powder are common intrawound prophylactic agents to prevent periprosthetic joint infection during primary total joint arthroplasty. The aims of this study are (1) to determine the minimal inhibitory concentration (MIC) and time to death for PI, CHG, and VANC against multiple bacteria and (2) to determine time to death against bacteria dried on titanium discs. Methods: A standard quantitative suspension assay was performed to determine the MIC for PI, CHG, and VANC against methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Haemophilus influenzae, Pseudomonas aeruginosa, Burkholderia cepacia, and Escherichia coli. Time to death assay was performed with time points of 0, 3, 30, and 60 minutes. Concentrations of antiseptic agents for time to death assay were 1% PI, 0.05% CHG, and 5 μg/mL VANC. Dry-phase bacteria on titanium discs were treated in a similar fashion. Results: The MIC of PI was 0.63%, CHG was 0.0031%, and VANC was 1.56 μg/mL. All 7 bacterial isolates were completely killed by PI at all times tested. CHG failed to kill MRSA and B cepacia at 0- and 3-minute exposures. Vancomycin completely killed MRSA and S epidermidis isolates between 18-20 hours of exposure. All bacterial isolates dried on titanium discs were eliminated by PI exposure on contact. E coli and S epidermidis were incompletely eliminated by CHG at 0 minutes, with all isolates eliminated at 3, 10, and 30 minutes. Conclusion: Our study suggests that PI kills all bacteria tested immediately on contact and that the exposure time is not the key factor.