We determined the effects of two antibiotic policies (predominance of either beta-lactam antibiotics or fluroquinolones) on acquisition with third-generation cephalosporin-resistant Enterobacteriaceae (CRE) and fluoroquinolone-resistant CRE (FCRE) in two ICUs, with monitoring of other variables that may influence acquisition. After an 8-month baseline period, units were randomized to a predominant beta-lactam antibiotic regimen (weekly cycling of ceftriaxone, amoxicillin-clavulanic acid and fluroquinolones) or a fluoroquinolone regimen for 3 months, with cross-over for another 3 months. Acquisition of CRE and FCRE was determined by microbiological surveillance. During baseline, acquisition rates for CRE and FCRE were 14/1,000 and 2/1,000 patient days at risk, respectively. Cross-transmission of CRE accounted for a parts per thousand currency sign25% of acquisitions, and CRE acquisition was associated with the use of beta-lactam antibiotics (amoxicillin-clavulanic acid in particular). As compared to baseline, beta-lactam antibiotic use [in defined daily dose (DDD)/1,000 patient days] was reduced from 854 to 526 (-39%) and 555 (-35%) during both intervention periods. Fluoroquinolone use was increased from 150 and 129 DDD/1,000 patient days in baseline and the beta-lactam period to 514 DDD/1,000 patient days (+243%) in the fluoroquinolone period. Reductions in beta-lactam use were not associated with reduced CRE acquisition [adjusted HRs were 1.0 (95% CR: 0.5-2.2) and 1.1 (95% CI: 0.5-2.5) during both periods, respectively]. Increased use of fluoroquinolones was associated with increased acquisition of FCRE [adjusted HR 4.1 (95% CI: 1.4-11.9; p < 0.01]. Infection control variables remained comparable during all periods. A 35-39% reduction of beta-lactam exposure was not associated with reduced acquisition of CRE, whereas a 243% increase of fluoroquinolone use increased acquisition of FCRE.