Resistance of bacteria to antibiotics is a global medical problem requiring close cooperation between veterinary and human physicians. Raw materials and foods of animal origin may be not only a source of pathogenic bacteria causing alimentary tract infections but also a source of bacteria with a dangerous extent of resistance to antibiotics, potentially entering the human food chain. This article presents results of the first study in the Czech Republic detecting the presence of Enterobacteriaceae-producing extended-spectrum b -lactamases (ESBLs) in swabs collected in slaughterhouses from surfaces of healthy animal carcasses. In 2012, swabs taken from pig (n = 166) and cattle (n = 140) carcass surfaces were analyzed. In 17 % of 53 studied slaughterhouses, ESBL-producing Escherichia coli strains were isolated. ESBLs were found in 11 and 4 % of porcine and bovine samples, respectively. Swabs collected from pigs yielded 18 ESBL-producing E. coli strains. The bla genes were found to encode production of CTX-M-1 group enzymes in 16 strains, SHV in one case, and both CTX-M-1-like and TEM in another case. In swabs taken from cattle, five ESBL-producing E. coli strains were isolated. In three cases, the bla genes for CTX-M-1-like production were identified; in two cases, genes for both CTX-M-1-like and TEM production were found. The similarity/identity of ESBL-positive isolates was compared by pulsed-field gel electrophoresis. This is the first report and characterization of the presence and nature of ESBL-producing E. coli in swabs collected from surfaces of healthy pig and cattle carcasses in slaughterhouses in the Czech Republic.