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Polymyxins are still widely used for the treatment of carbapenem-resistant <i>Acinetobacter baumannii</i> and <i>Pseudomonas aeruginosa</i> bloodstream infections (BSIs). This study seeks to evaluate the impact of polymyxin B versus colistin on mortality and nephrotoxicity in BSI caused by these bacteria. We conducted a retrospective cohort study from 2014 to 2021 in Porto Alegre, Brazil. We included patients aged ≥18 years and excluded patients with polymicrobial infection or treatment for ≤48 h. The 30-day mortality was the primary outcome evaluated through Cox regression. We included 259 patients with BSI episodes: 78.8% caused by <i>A. baumannii</i> and 21.2% caused by <i>P. aeruginosa</i>. Polymyxin B did not impact mortality compared to colistin (adjusted hazard ratio (aHR), 0.82; 95% confidence interval (CI), 0.52–1.30; <i>p</i> = 0.40 (when adjusted for COVID-19 comorbidity, <i>p</i> = 0.05), Pitt bacteremia score, <i>p</i> < 0.01; Charlson comorbidity index, <i>p</i> < 0.001; time to start active antimicrobial therapy, <i>p</i> = 0.02). Results were maintained in the subgroups of BSI caused by <i>A. baumannii</i> (aHR, 0.92; 95% CI, 0.55–1.54; <i>p</i> = 0.74), <i>P. aeruginosa</i> (aHR, 0.47; 95% CI, 0.17–1.32; <i>p</i> = 0.15) and critical care patients (aHR, 0.77; 95% CI, 0.47–1.26; <i>p</i> = 0.30). Treatment with polymyxin B or colistin did not impact 30-day mortality in patients with carbapenem-resistant <i>A. baumannii</i> or <i>P. aeruginosa</i> BSI.