BACKGROUND: Mecillinam (amdinocillin) is active against Gram-negative bacteria. Clinical data on the efficacy of IV mecillinam for severe urinary tract infections is sparse. OBJECTIVES: To assess the effectiveness of targeted IV mecillinam compared with other β-lactams for bacteraemia with Escherichia coli and Klebsiella spp. and a urinary tract focus. PATIENTS AND METHODS: We performed a retrospective cohort study at five university hospitals in the Capital Region of Denmark from 1 January 2012 to 31 December 2017. We used Cox proportional hazard regression to compare the primary composite endpoint (all-cause mortality or bacteraemia recurrence within 30 days) between patients treated with mecillinam versus ampicillin, cefuroxime, piperacillin/tazobactam and meropenem. RESULTS: We included 1129 patients in the primary analysis, of which 146 were given IV mecillinam as targeted treatment. We found no significant difference in the primary endpoint between patients treated with mecillinam versus ampicillin and cefuroxime, but found a higher risk for the primary endpoint in the piperacillin/tazobactam and meropenem groups, with adjusted HRs of 2.22 (95% CI 1.24-3.97, P < 0.01) and 2.48 (95% CI 1.04-5.93, P = 0.04), respectively, compared with mecillinam. CONCLUSIONS: The results of this study suggest that IV mecillinam may be a suitable targeted treatment for bacteraemia with a urinary tract focus. However, these results need confirmation by randomized controlled studies.