Background: Over the past decade, the CLSI has updated susceptibility break points for several antimicrobial agents. The purpose of this study was to evaluate the impact of these changes against gram-negative bacteria at our academic medical center. Methods: In this retrospective, IRB-approved study, we collected consecutive, nonduplicate clinical isolates of Enterobacter cloacae, Escherichia coli, Klebsiella aerogenes, K. oxytoca, K. pneumoniae, and Pseudomonas aeruginosa for the past decade (2010–2019) at our academic medical center and 3 adult ICUs. Susceptibility testing was performed using the BD Phoenix automated system. For these isolates, susceptibilities for 7 β-lactams (aztreonam, ceftriaxone, ceftazidime, cefepime, piperacillin/tazobactam, ertapenem, and meropenem) and 2 fluoroquinolones (levofloxacin, ciprofloxacin) were calculated based upon CLSI break points in 2010 and current CLSI break points in 2020. Any change >5% in susceptibility was deemed significant for this analysis. Results: In 17.5% of Enterobacteriales isolates tested, at least 1 antimicrobial demonstrated significant decline. Ertapenem was the most commonly affected antimicrobial (45% of the isolates) followed by ceftriaxone (35%) and cefepime (25%). Susceptibilities of aztreonam, ceftazidime, and meropenem were not affected for any of the Enterobacteriales. The most common organism demonstrating a significant impact on change in susceptibility among the Enterobacteriales was E. cloacae (41.7% of the time) followed by E. aerogenes (20.8%), K. oxytoca (12.5%), K. pneumoniae (8.3%) and E. coli (4.2%). Most of the impact was observed hospital-wide (33.3%), followed closely by the MICU (28.6%), the NSICU (23.8%) and the CVICU (14.3%). For P. aeruginosa, the impact of the antimicrobial break-point changes on susceptibility was more pronounced than the Enterobacteriales. Overall, 93.8% of the time there was a significant decline in antimicrobial susceptibility. Each antimicrobial (ciprofloxacin, levofloxacin, meropenem, and piperacillin/tazobactam) demonstrated a significant decline in susceptibility hospital-wide and in each ICU except for the susceptibility of meropenem in the NSICU. Conclusions: Changes in break points had a significant impact on the susceptibility of all antimicrobials for P. aeruginosa at our institution, both hospital-wide and in the adult ICUs. Although the impact was less for the Enterobacteriales, ertapenem, ceftriaxone, and cefepime demonstrated significant susceptibility changes, especially with E. cloacae. Understanding and evaluating the impact of the break-point changes may lead to changes in empiric therapy in other institutions.