The aim of this study was to determine the prevalence of vaginal Escherichia coli colonization and perianal carriage of Enterobacteriaceae resistant to third generation cephalosporins in pregnant women. Vaginal and perianal samples from 259 pregnant women were studied. Vaginal swabs were inoculated onto MacConkey agar plates and perianal swabs were inoculated onto CHROMagar extended-spectrum beta-lactamase (ESBL) plates. The minimal inhibitory concentration of the isolates was determined using the Epsilometer test method. The phenotypic detection of ESBLs was performed by the combined disc method using cefotaxime versus cefotaxime plus clavulanate. The prevalence of vaginal E. coli colonization during pregnancy was 14.3%. The resistance rate to ampicillin, gentamicin, and cefotaxime was 48.6, 10.8, and 0.8%, respectively. Enterobacteriaceae resistant to third generation cephalosporins were recovered in 7.3% of all perianal specimens. Among them, 5.4% of pregnant women were colonized with E. coli ESBL-producer strains. The present study revealed that colonization with Enterobacteriaceae resistant to third generation cephalosporins is significant in pregnancy. ESBL-producing E. coli were the most prevalent organisms. Screening strategies designed to monitor for ESBL-producing E. coli could be useful in endemic areas to prevent perinatal transmission and the introduction of multiresistant strains to the maternity ward.