Background: The dramatic spread of vancomycin-resistant enterococci (VRE) among hemodialysis (HD) patients led to the replacement of vancomycin with cephalosporins as part of the primary empiric therapy for bacterial infections in HD units. The aim of the study was to examine the effects of this new regimen on the colonization rate of resistant bacteria among HD patients. Methods: Rectal swabs were taken from 105 HD patients and 91 control hospitalized patients. Groups were matched for age, sex, nursing home residency and background diseases. Enterococci were tested for vancomycin resistance, Staphylococcus aureus isolates were tested for methicillin resistance (MRSA), and Enterobacteriaceae were tested for extended-spectrum β-lactamase (ESBL) activity. Results: In the HD group 1 VRE, 1 MRSA and 9 ESBL-producing organisms were isolated compared to 1 MRSA and 1 ESBL organism in the control group (p = 0.018 for ESBL). In the year prior to the study, the use of cephalosporins had been enhanced in the HD group compared to the control group (p < 0.001), and in the HD ESBL-positive patients compared to the HD ESBL-negative ones (p = 0.007). The overall use of antibiotics in the control group was the same as in the HD group. In a subanalysis of the HD group alone, the ESBL carriers were older, sicker, used more antibiotics, were hospitalized frequently and had a higher mortality rate, compared to noncarriers. Conclusions: The use of cephalosporins as first-line therapy in HD patients in central Israel reduced the prevalence of VRE colonization but may have contributed to the emergence of ESBL-producing organisms through induction of selection pressure. This may lead to serious complications in the management of these patients.