Chlorhexidine bathing to prevent transmission of multidrug-resistant organisms has been adopted by many U.S. hospitals, but increasing chlorhexidine use has raised concerns about possible emergence of resistance. We sought to establish a broth microdilution method for determining chlorhexidine MICs and then used the method to evaluate chlorhexidine MICs for bacteria that can cause health care-associated infections. We adapted a broth microdilution method for determining chlorhexidine MICs, poured panels, established quality control ranges, and tested Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae complex isolates collected at three U.S. sites. Chlorhexidine MICs were determined for 535 isolates including 129 S. aureus, 156 E. coli, 142 K. pneumoniae, and 108 E. cloacae complex isolates. The respective MIC distributions for each species ranged from 1 to 8 mg/L (MIC50 = 2 mg/L and MIC90 = 4 mg/L), 1 to 64 mg/L (MIC50 = 2 mg/L and MIC90 = 4 mg/ L), 4 to 64 mg/L (MIC50 = 16 mg/L and MIC90 = 32 mg/L), and 1 to .64 mg/L (MIC50 = 16 mg/L and MIC90 = 64 mg/L). We successfully adapted a broth microdilution procedure that several laboratories were able to use to determine the chlorhexidine MICs of bacterial isolates. This method could be used to investigate whether chlorhexidine MICs are increasing. IMPORTANCE Chlorhexidine bathing to prevent transmission of multidrug-resistant organisms and reduce health care-associated infections has been adopted by many hospitals. There is concern about the possible unintended consequences of using this agent widely. One possible unintended consequence is decreased susceptibility to chlorhexidine, but there are not readily available methods to perform this evaluation. We developed a method for chlorhexidine MIC testing that can be used to evaluate for possible unintended consequences.