OBJECTIVES: This study aimed to describe the epidemiology and antimicrobial susceptibility patterns of gram-negative pathogens in Brazil from 2018 to 2020, addressing the gap in national data on healthcare-associated infections, using information from a private laboratory network. METHODS: A cross-sectional study was conducted using a database from Fleury hospital network, a private laboratory in Brazil. The analysis included blood, urine, and lower respiratory tract samples collected from January 2018 to June 2020. The study included consecutive non-duplicated isolates of Enterobacterales or P. aeruginosa from inpatients aged ≥18 years old. Bacterial identification was performed using mass spectrometry, and antimicrobial susceptibility was determined following EUCAST/BrCAST guidelines. RESULTS: A total of 25,180 isolates were included in the analysis. Most of the sample consisted of female patients (57.9%), with a mean age of 70 years (SD 18.1). Enterobacterales were the most prevalent pathogens found (76.2%), while P. aeruginosa was present in the remaining 23.8%. In terms of antimicrobial susceptibility, Enterobacterales exhibited a higher susceptibility rate to ceftazidime/avibactam (97.1%) and amikacin (95.6%), while P. aeruginosa showed a higher susceptibility rate to polymyxin B (97.1%) and ceftolozane/tazobactam (86.6%). Among carbapenem-resistant P. aeruginosa isolates, 75% were susceptible to ceftolozane/tazobactam. Additionally, 24.2% of K. pneumoniae complex samples were identified. CONCLUSION: Enterobacterales was the most frequently encountered group in Brazil. Ceftazidime/avibactam and amikacin demonstrated the highest efficacy against this group, while ceftolozane/tazobactam and polymyxin had the highest efficacy against P. aeruginosa. This highlights the importance of new β-lactam-β-lactamase inhibitor combinations for the treatment of gram-negative infections.