Summary Background Antimicrobial stewardship is advocated to improve the quality of antimicrobial use. We did a systematic review and meta-analysis to assess whether antimicrobial stewardship objectives had any effects in hospitals and long-term care facilities on four predefined patients' outcomes: clinical outcomes, adverse events, costs, and bacterial resistance rates. Methods We identified 14 stewardship objectives and did a separate systematic search for articles relating to each one in Embase, Ovid MEDLINE, and PubMed. Studies were included if they reported data on any of the four predefined outcomes in patients in whom the specific antimicrobial stewardship objective was assessed and compared the findings in patients in whom the objective was or was not met. We used a random-effects model to calculate relative risk reductions with relative risks and 95% CIs. Findings We identified 145 unique studies with data on nine stewardship objectives. Overall, the quality of evidence was generally low and heterogeneity between studies was mostly moderate to high. For the objectives empirical therapy according to guidelines, de-escalation of therapy, switch from intravenous to oral treatment, therapeutic drug monitoring, use of a list of restricted antibiotics, and bedside consultation the overall evidence showed significant benefits for one or more of the four outcomes. Guideline-adherent empirical therapy was associated with a relative risk reduction for mortality of 35% (relative risk 0·65, 95% CI 0·54–0·80, p<0·0001) and for de-escalation of 56% (0·44, 0·30–0·66, p<0·0001). Evidence of effects was less clear for adjusting therapy according to renal function, discontinuing therapy based on lack of clinical or microbiological evidence of infection, and having a local antibiotic guide. We found no reports for the remaining five stewardship objectives or for long-term care facilities. Interpretation Our findings of beneficial effects on outcomes with nine antimicrobial stewardship objectives suggest they can guide stewardship teams in their efforts to improve the quality of antibiotic use in hospitals. Funding Dutch Working Party on Antibiotic Policy and Netherlands National Institute for Public Health and the Environment.