Sepsis begins outside of the hospital for nearly 80% of patients and the emergency room (ER) represents the first contact with the health care system. This study evaluates a project to improve collection of blood cultures (BCs) in patients with sepsis in the ER consisting of staff education and completion of the appropriate BC pre-analytical phase. A retrospective observational study performed to analyse the data on BC collection in the ER before and after a three-phase project. The first phase (1 January to 30 June 2015) before the intervention consisted of evaluation of data on BCs routinely collected in the ER. The second phase (1 July to 31 December 2015) was the intervention phase in which educational courses on sepsis recognition and on pre-analytical phase procedures (including direct incubation) were provided to ER staff. The third phase (1 January to 30 June 2016; after the intervention) again consisted of evaluation. Before the intervention, out of 24,738 admissions to the ER, 103 patients (0.4%) were identified as septic and had BCs drawn (359 BC bottles); 19 out of 103 patients (18.4%) had positive BCs. After the intervention, out of 24,702 admissions, 313 patients (1.3%) had BCs drawn (1,242 bottles); of these, 96 (30.7%) had positive BCs. Comparing the first and third periods, an increase in the percentage of patients with BCs collected (from 0.4% to 1.3% respectively, p < 0.0001) and an increase in the percentages of patients with true-positive BCs (from 0.08% to 0.39% of all patients evaluated respectively, p < 0.0001) were observed. The isolation of bacteria by BCs increased 3.25-fold after project implementation. These results can be principally ascribed to an improved awareness of sepsis in the staff associated with improved pre-analytical phase procedures in BC collection.