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•The burden of invasive ExPEC infections is increasing.•A glycoconjugate vaccine, containing ten E. coli O-antigens, is in development.•Vaccine effectiveness will depend on serogroup distribution.•Common serogroups in ICU patients with invasive ExPEC were O25, O8, O2, O6, and O15.•Theoretical vaccine coverage was 54% in this population. Invasive infections by extra-intestinal pathogenic Escherichia coli (ExPEC) strains are increasing. We determined O-serogroups of E. coli isolates from ICU patients having bloodstream infections (BSI) and the potential coverage of a 10-valent O-polysaccharide conjugate vaccine currently in development for the prevention of invasive ExPEC disease. We studied E. coli BSI among patients admitted to a tertiary ICU in the Netherlands between April 2011 and November 2016. O-serogroups were determined in vitro by agglutination and whole genome sequencing. Among 714 ICU patients having BSI, 70 (10%) had an E. coli BSI. Among 68 (97%) isolates serogrouped, the most common serogroups were O25 (n = 11; 16%), O8 (n = 5; 7%), O2 (n = 4; 6%), O6 (n = 4; 6%), and O15 (n = 4; 6%). The theoretical coverage of a 10-valent ExPEC vaccine was 54% (n = 37). A multi-valent ExPEC O-polysaccharide conjugate vaccine in development could potentially aid in the prevention of E. coli BSI in Dutch ICU patients.