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Objectives To explore the association between MIC/EUCAST breakpoint ratio and 28 day mortality in patients with a Gram-negative bloodstream infection (BSI). Methods Using data from the Bloodstream Infection—Focus on Outcomes (BSI-FOO) observational study, we defined an average MIC/EUCAST breakpoint ratio that was updated daily to reflect changes in treatment in the first 7 days after blood culture. Cox regression analysis was performed to estimate the association between MIC/EUCAST breakpoint ratio and mortality, adjusting for organism and a risk score calculated using potential confounding variables. The primary outcome was 28 day all-cause mortality from the date of blood culture. Results Of the 1903 study participants, 514 met the eligibility criteria and were included in the analysis (n = 357 Escherichia coli, n = 6 Klebsiella and n = 151 Pseudomonas aeruginosa). The average age was 74.0 years (IQR 60.0–82.0). The mortality rate varied from 11.1% (in patients treated with an average MIC/EUCAST breakpoint ratio of 1) to 27.6% (in patients treated with antibiotics with an average MIC/EUCAST breakpoint ratio >1). After adjusting for risk score and organism, MIC/EUCAST breakpoint ratio was not associated with 28 day mortality (P = 0.148). Conclusions In an adjusted model controlling for potential confounding variables, there was no evidence to suggest a relationship between MIC/EUCAST breakpoint ratio and 28 day mortality in patients with a Gram-negative BSI.