Our goal was to determine the characteristics and the mode of acquisition of healthcare-associated bacteraemia due to CTX-M-producing Escherichia coli in a 1,800-bed hospital. Sixteen extended-spectrum beta-lactamase (ESBL)-producing E. coli strains were collected between 2001 and 2006 from patients with bloodstream infections. The incidence density of these infections increased from 0.002 to 0.02 per 1,000 days of hospitalisation during the study period. Most of the strains (87%) produced a CTX-M-type enzyme associated with TEM-1 (86%), OXA-30 (50%), AAC(3)-II (57%), AAC(6') (50%) and QnrS1 (7%). When present (n = 8), the bla (CTX-M-15) gene was always located downstream of the insertion sequence ISEcp1. Co-resistance was generally observed: fluoroquinolones (81%), trimethoprim-sulfamethoxazole (62%) and/or aminoglycosides (69%). Although the strains were found to be genetically unrelated, most of the cases were hospital-acquired (69%) or healthcare-associated (25%), underlining the need for infection control measures to limit the spread of ESBL-producing E. coli in hospital settings.