The emerging resistance to cephalosporins and carbapenems in gram-negative pathogens poses significant health challenges and increased treatment failures. The development and evaluation of novel therapeutic options are needed urgently. This study aimed to assess the in vitro activity of ceftolozane/tazobactam (C/T), a new cephalosporin and β-lactamase inhibitor combination, against isolates of Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae from different infection sources in two general hospitals in Singapore. Susceptibility testing revealed that C/T has good activity against 600 tested gram-negative pathogens, showing a 94.8% susceptibility rate across different species and infection types. Additionally, emergence of C/T resistance was also observed and characterized with whole genome sequencing (WGS) analysis. WGS analysis indicated that C/T resistance was predominantly associated with harbouring broad-spectrum β-lactamases such as carbapenemases, ESBLs, AmpC and others. Novel mutations in penicillin-binding-protein 3 (PBP3), the target of ceftolozane, and variants associated with hyper ampC expression (ampD, ampR, and dacB), were also found in C/T-resistant isolates, potentially contributing to C/T resistance. The finding of this study provides valuable local data on C/T susceptibility, which is important for guiding the proper clinical use of C/T and promoting antimicrobial stewardship in managing Gram-negative infections.