Surveillance of antimicrobial resistance among gram-negative bacteria (GNB) is of critical importance, but data for Peru are not available. To fill this gap, a non-interventional hospital-based surveillance study was conducted in 15 hospitals across Peru from July 2017 to October 2019. Consecutive unique blood culture isolates of key GNB (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter spp.) recovered from hospitalized patients were collected for centralized antimicrobial susceptibility testing, along with linked epidemiological and clinical data. A total of 449 isolates were included in the analysis. Resistance to third-generation cephalosporins (3GCs) was present in 266 (59.2%) GNB isolates. Among E. coli (n = 199), 68.3% showed 3GC resistance (i.e., above the median ratio for low- and middle-income countries in 2020 for this sustainable development goal indicator). Carbapenem resistance was present in 74 (16.5%) GNB isolates, with wide variation among species (0% in E. coli, 11.0% in K. pneumoniae, 37.0% in P. aeruginosa, and 60.8% in Acinetobacter spp. isolates). Co-resistance to carbapenems and colistin was found in seven (1.6%) GNB isolates. Empiric treatment covered the causative GNB in 63.3% of 215 cases. The in-hospital case fatality ratio was 33.3% (92/276). Pseudomonas aeruginosa species and carbapenem resistance were associated with higher risk of in-hospital death. In conclusion, an important proportion of bloodstream infections in Peru are caused by highly resistant GNB and are associated with high in-hospital mortality.