A prospective cohort study was performed among travelers from the Netherlands to investigate the acquisition of carbapenemase-producing Enterobacteriaceae (CP-E) and extended-spectrum β-lactamase–producing Enterobacteriaceae (ESBL-E) and associated risk factors. Questionnaires were administered and rectal swabs were collected and tested before and after return. Of 370 travelers, 32 (8.6%) were colonized with ESBL-E before travel; 113 (30.5%) acquired an ESBL-E during travel, and 26 were still colonized 6 months after return. No CP-E were found. Independent risk factors for ESBL-E acquisition were travel to South and East Asia. Multilocus sequence typing showed extensive genetic diversity among Escherichia coli. Predominant ESBLs were CTX-M enzymes. The acquisition rate, 30.5%, of ESBL-E in travelers from the Netherlands to all destinations studied was high. Active surveillance for ESBL-E and CP-E and contact isolation precautions may be recommended at admission to medical facilities for patients who traveled to Asia during the previous 6 months.