Objective To determine if the introduction of an emergency department (ED) sepsis screening tool and management bundle affects antibiotic prescribing and use. Design Multicentre, cohort, before-and-after study design. Setting Three tertiary hospitals in Queensland, Australia (median bed size 543, range 520–742). Participants Adult patients, presenting to the ED with symptoms and signs suggestive of sepsis who had blood cultures collected. These participants were further assessed and stratified as having septic shock, sepsis or infection alone, using Sepsis-3 definitions. The study dates were 1 July 2017–31 March 2020. Intervention The breakthrough series collaborative ‘Could this be Sepsis?’ Programme, aimed at embedding a sepsis screening tool and treatment bundle with weighted-incidence syndromic combined antibiogram-derived antibiotic guidelines in EDs. Main outcome measures The primary outcome was the rate of empirical prescriptions adherent to antibiotic guidelines during the ED encounter. Secondary outcomes included the empirical prescriptions considered appropriate, effective antibiotics administered within 3 hours and assessment of harm measures. Results Of 2591 eligible patients, 721 were randomly selected: 241 in the baseline phase and 480 in the post-intervention phase. The rates of guideline adherence were 54.0% and 59.5%, respectively (adjusted OR (aOR) 1.41 (95% CI 1.00, 1.98)). As compared with baseline, there was an increase in the rates of appropriate antibiotic prescription after bundle implementation (69.9% vs 57.1%, aOR 1.92 (95% CI 1.37, 2.68)). There were no differences between the baseline and post-intervention groups with respect to time to effective antibiotics, adverse effects or ED rates of broad-spectrum antibiotic use. Conclusion and relevance The use of an ED sepsis screening tool and management bundle was associated with an improvement in the rates of appropriate antibiotic prescription without evidence of adverse effects.