© 2016 The Authors.Unlike Escherichia coli strains belonging to phylogroup B2, the clinical significance of strains belonging to phylogroup F is not well understood. Here we report on a collection of phylogroup F strains recovered in Australia from faeces and extra-intestinal sites from humans, companion animals and native animals, as well as from poultry meat and water samples. The distribution of sequence types was clearly non-random with respect to isolate source. The antimicrobial resistance and virulence trait profiles also varied with the sequence type of the isolate. Phylogroup F strains tended to lack the virulence traits typically associated with phylogroup B2 strains responsible for extra-intestinal infection in humans. Resistance to fluoroquinolones and/or expanded-spectrum cephalosporins was common within ST648, ST354 and ST3711. Although ST354 and ST3711 are part of the same clonal complex, the ST3711 isolates were only recovered from native birds being cared for in a single wildlife rehabilitation centre, whereas the ST354 isolates were from faeces and extra-intestinal sites of dogs and humans, as well as from poultry meat. Although ST354 isolates from chicken meat in Western Australia were distinct from all other ST354 isolates, those from poultry meat samples collected in eastern Australia shared many similarities with other ST354 isolates from humans and companion animals.