Introduction: In view of the continuous rise in Gram-negative bacterial resistance and limited treatment options, Ceftolozane/tazobactam (C/T) is a newly introduced antimicrobial agent in Lebanon for its demonstrated activity against resistant Gram-negative bacteria. However, in vitro data is not available about its activity against commonly isolated bacteria in this country. Methodology: The analysis included clinical isolates, multidrug–resistant (MDR) and extended-spectrum Beta-lactamases (ESBLs), representing 124 Escherichia coli, 75 Klebsiella pneumoniae and 100 Pseudomonas aeruginosa, identified using the MALDI-TOF. The minimum inhibitory concentration (MIC) for C/T was determined by the Etest (Liofilchem, Roseto degli Abruzzi, Italy). In addition, the disk diffusion (DD) test was used to determine the activity of C/T and of the antimicrobials routinely used to test for such pathogens. Results: The C/T activity against the ESBL producers E. coli and K. pneumoniae isolates were similar (MIC90 value of 1 and 1.5 µg/mL, respectively; susceptibility of 100% and 96%, respectively). However, the activity of C/T against the E. coli and K. pneumoniae MDR isolates was much lower (MIC90 value of 256 and 96 µg/mL, respectively; susceptibility of 54% for each). The C/T MIC90 value for the non-MDR P. aeruginosa isolates was 3 µg/mL and ≥ 256 µg/mL for the MDR P. aeruginosa isolates (susceptibility of 96% vs 42% respectively). Overall, the C/T activities show comparable or higher susceptibility to the routinely used antimicrobials. Conclusion: The high in vitro activity of C/T points out its value as a possible alternative to the antimicrobials currently used for treatment of infections caused by such pathogens and would help in minimizing toxicity and bacterial resistance.