Non-adherence to antimicrobial guidelines in patients with bloodstream infection can result in undertreatment, overtreatment, or equivalent treatment, and could lead to suboptimal care. Our aim was to examine the association between non-adherence and appropriate coverage as well as to assess the impact of nonadherence on 30-day mortality. Methods: We conducted a retrospective cohort study between 2012 and 2017 at a tertiary university hospital. Adult patients attending the emergency department with a bloodstream infection were included. Adherence was defined as guideline-recommended antibiotic therapy. Non-adherence was either undertreatment (too narrowspectrum), overtreatment (too broad-spectrum), or equivalent treatment. Outcomes were appropriate coverage (i.e. antibiotic therapy that matches in vitro susceptibility of the isolated bacteria) and 30-day mortality. Results: We included 909 patients of whom 395 (43.5%) were treated adherently, 355 (39.1%) were undertreated, 87 (9.6%) were overtreated, and 72 (7.9%) received an equivalent treatment. Overtreated patients were more severely ill, whilst undertreated patients had more favorable patient characteristics. Overtreatment did not result in higher appropriate coverage, whereas undertreatment was associated with lower coverage (OR[95%CI]: 0.18 [0.12; 0.26]). Overtreatment and undertreatment were not associated with 30-day mortality. Conclusions: Guideline adherence likely depends on disease severity, because overtreatment was more often observed in patients with high disease severity and undertreatment in less severely ill patients. Undertreatment was associated lower appropriate coverage but not with higher mortality. However, this can be the result of residual confounding . Overtreatment did not result in higher appropriate antibiotic coverage nor a survival benefit . Therefore, overtreatment seems not justifiable.