Objectives Antibiotic resistance has risen dramatically over the past years. For individual patients, adequate initial antibiotic therapy is essential for clinical outcome. Computer-assisted decision support systems (CDSSs) are advocated to support implementation of rational anti-infective treatment strategies based on guidelines. The aim of this study was to evaluate long- term effects after implementation of a CDSS. Design This prospective ‘before/after’ cohort study was conducted over four observation periods within 5 years. One preinterventional period (pre) was compared with three postinterventional periods: directly after intensive implementation efforts (post1), 2 years (post2) and 3 years (post3) after implementation. Setting Five anaesthesiological-managed intensive care units (ICU) (one cardiosurgical, one neurosurgical, two interdisciplinary and one intermediate care) at a university hospital. Participants Adult patients with an ICU stay of >48 h were included in the analysis. 1316 patients were included in the analysis for a total of 12 965 ICU days. Intervention Implementation of a CDSS. Outcome measures The primary end point was percentage of days with guideline adherence during ICU treatment. Secondary end points were antibiotic-free days and all-cause mortality compared for patients with low versus high guideline adherence. Main results Adherence to guidelines increased from 61% prior to implementation to 92% in post1, decreased in post2 to 76% and remained significantly higher compared with baseline in post3, with 71% (p=0.178). Additionally, antibiotic-free days increased over study periods. At all time periods, mortality for patients with low guideline adherence was higher with 12.3% versus 8% (p=0.014) and an adjusted OR of 1.56 (95% CI 1.05 to 2.31). Conclusions Implementation of computerised regional adapted guidelines for antibiotic therapy is paralleled with improved adherence. Even without further measures, adherence stayed high for a longer period and was paralleled by reduced antibiotic exposure. Improved guideline adherence was associated with reduced ICU mortality.