BACKGROUND: Extended-spectrum β-lactamase (ESBL)-producing Enterobacterales (ESBL-PE) are highly prevalent in long-term care (LTCF) settings. In order to estimate the acquisition rate of ESBL-producing Escherichia coli and Klebsiella pneumoniae in LTCF settings, and identify clinical and environmental risk factors, a multi-centre, prospective cohort study was conducted in six LTCFs in Germany, France, Spain and the Netherlands. METHODS: Longitudinal screening of residents was performed over 32 weeks, collecting epidemiological and clinical data and environmental samples. The primary outcome was the rate of new acquisition of ESBL-PE among LTCF residents. Molecular epidemiology was studied using whole genome sequencing, and risk factor analysis was undertaken using logistic and Poisson regression models. RESULTS: In total, 299 residents provided 1958 samples during follow-up. The prevalence of ESBL-PE colonization at baseline was 16.4%, and the incidence of acquisition was 0.79 per 1000 resident-days, both with high variability between LTCFs. Age ≥80 years, vascular disease and antibiotic consumption within the preceding year were risk factors for baseline colonization. Lack of hand sanitizers and a low nurse:resident ratio were associated with colonization. The presence of medical devices was associated with risk of acquisition. Vascular disease, hemiplegia, antibiotic consumption, and non-availability of private bathrooms were associated with carriage of multiple sequence types (STs). The prevalence of ESBL-PE among environmental samples was 2%, exclusively in LTCFs with high prevalence among residents. Genetic analysis showed a high prevalence of ST10 E. coli and ST405 K. pneumoniae at two study sites. CONCLUSION: Infection prevention interventions, including availability of hand sanitizers, the number of nurses per resident, and antimicrobial stewardship, constitute important measures to control ESBL-PE in LTCFs. Genome-based surveillance could guide targeted interventions.