Escherichia coli isolates of serotype O18:K1:H7, taken from women with acute cystitis, healthy control patients, and infants with neonatal bacterial meningitis (NBM), were analyzed and were compared with phylogenetically diverse control strains from the E. coli Reference collection. Clonal relationships were defined by amplification phylotyping, nicotinamide auxotrophy, and outer membrane protein patterns (OMPs). Virulence factor profiles were determined by multiplex polymerase chain reaction, probe hybridization, and hemagglutination testing. The O18:K1:H7 cystitis, fecal, and NBM isolates were clonally derived. The cystitis isolates and archetypal NBM isolates RS218 and C5 were from the OMP6 subclone of E. coli O18:K1:H7 and exhibited a consensus virulence genotype that included papG allele III (cystitis-associated P fimbrial adhesin), sfaS (S fimbrial adhesin), hlyA (hemolysin), cnf1 (cytotoxic necrotizing factor), iroN (putative siderophore), and ibeA (invasion of brain endothelium). The demonstrated commonality between O18:K1:H7 isolates from cystitis and NBM suggests common pathogenetic mechanisms and the possibility of new approaches to prevention.