Background: Chlorhexidine bathing reduces bacterial skin colonization and prevents infections in specific patient populations. As chlorhexidine use becomes more widespread, concerns about bacterial tolerance to chlorhexidine have increased; however, testing for chlorhexidine minimum inhibitory concentrations (MICs) is challenging. We adapted a broth microdilution (BMD) method to determine whether chlorhexidine MICs changed over time among 4 important healthcare-associated pathogens. Methods: Antibiotic-resistant bacterial isolates (Staphylococcus aureus from 2005 to 2019 and Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae complex from 2011 to 2019) were collected through Emerging Infections Program surveillance in 2 sites (Georgia and Tennessee) or through public health reporting in 1 site (Orange County, California). A convenience sample of isolates were collected from facilities with varying amounts of chlorhexidine use. We performed BMD testing using laboratory-developed panels with chlorhexidine digluconate concentrations ranging from 0.125 to 64 μg/mL. After successfully establishing reproducibility with quality control organisms, 3 laboratories performed MIC testing. For each organism, epidemiological cutoff values (ECVs) were established using ECOFFinder. Results: Among 538 isolates tested (129 S. aureus, 158 E. coli, 142 K. pneumoniae, and 109 E. cloacae complex), S. aureus, E. coli, K. pneumoniae, and E. cloacae complex ECVs were 8, 4, 64, and 64 µg/mL, respectively (Table 1). Moreover, 14 isolates had an MIC above the ECV (12 E. coli and 2 E. cloacae complex). The MIC50 of each species is reported over time (Table 2). Conclusions: Using an adapted BMD method, we found that chlorhexidine MICs did not increase over time among a limited sample of S. aureus, E. coli, K. pneumoniae, and E. cloacae complex isolates. Although these results are reassuring, continued surveillance for elevated chlorhexidine MICs in isolates from patients with well-characterized chlorhexidine exposure is needed as chlorhexidine use increases.