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Studies examining time to positivity (TTP) of blood cultures as a risk factor for death have shown conflicting results. The study objective was to examine the effect of TTP on all-cause-30-day case-fatality among a population-based cohort of patients with bloodstream infections (BSI). A retrospective cohort study including all residents of Queensland, Australia with incident monomicrobial BSI managed in the publicly funded healthcare system from 2000 to 2019 was performed. Clinical, TTP and all-cause 30-day case-fatality information was obtained from state-wide sources. A cohort of 88 314 patients was assembled. The median TTP was 14 hours, with 5th, 25th, 75th, and 95th percentiles of 4, 10, 20, and 53 hours, respectively. The TTP varied significantly by BSI aetiology. The 30-day all-cause case-fatality rate was 2606/17 879 (14.6%), 2834/24 272 (11.7%), 2378/20 359 (11.7%), and 2752/22 431 (12.3%) within the first, second, third, and fourth TTP quartiles, respectively (p < 0.0001). After adjustment for age, sex, onset, comorbidity, and focus of infection, TTP within 10 hours (first quartile) was associated with a significantly increased risk for death (odds ratio 1.43; 95% CI, 1.35–1.50; p < 0.001). After adjustment for confounding variables (odds ratio; 95% CI), TTP within the first quartile for Staphylococcus aureus (1.56; 1.41–1.73), Streptococcus pneumoniae (1.91; 1.49–2.46), β-hemolytic streptococci (1.23; 1.00–1.50), Pseudomonas species (2.23; 1.85–2.69), Escherichia coli (1.37; 1.23–1.53), Enterobacterales (1.38; 1.16–1.63), other Gram-negatives (1.68; 1.36–2.06), and anaerobes (1.58; 1.28–1.94) increased the risk for case-fatality. This population-based analysis provides evidence that TTP is an important determinant of mortality among patients with BSI. [ABSTRACT FROM AUTHOR]