Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs) in adult women, which are increasingly refractory to antimicrobial treatment. UPEC colonizes the vagina prior to causing a UTI. Our hypothesis was that the vaginal flora would be enriched in UPEC and therefore have a greater prevalence of non-susceptibility relative to the rectal flora. We used disk diffusion to determine the antimicrobial susceptibilities of 100 cervico-vaginal E. coli (CVEC) and 100 rectal E. coli (REC) isolates from 200 different patients. Phylogeny, plasmid replicons, and antimicrobial resistance genes were detected by polymerase chain reaction (PCR). There were no significant differences between CVEC and REC, and the overall levels of non-susceptibility were 39.5% for ampicillin (AM), 11.5% for ampicillin-sulbactam (SAM), 11.5% for trimethoprim-sulfamethoxazole (SXT), 5% for ciprofloxacin (CIP), 2.5% for nitrofurantoin (F/M), 0.5% for ceftazidime (CAZ), 0.5% for cefotaxime (CTX), and 0% for fosfomycin (FOS). SXT non-susceptibility was associated with phylogenetic groups A and D compared with B2. AM and SXT non-susceptibility was associated with plasmid carriage. The vaginal flora is not enriched in antimicrobial non-susceptibility relative to the rectal flora. However, antimicrobial non-susceptibility was associated with phylogeny and plasmid carriage.