The study aimed to determine the occurrence of Enterobacteriaceae producing broad-spectrum beta-lactamases, vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus strains in poultry in Moravia, Czech Republic, including phenotypic and genotypic analyses of the extent of resistance. Using chromogenic screening media, a total of 240 clinical samples collected from poultry and the poultry farm environment were processed. Phenotypic tests identified 23 isolates of broad-spectrum beta-lactamase-producing Escherichia coli and one VRE isolate (Enterococcus faecium with VanA resistance). Methicillin-resistant Staphylococcus aureus strains were not detected. Among the isolates producing broad-spectrum beta-lactamases, 17 produced extended-spectrum beta-lactamases, most frequently CTX-M; the remaining 6 isolates were CIT-type AmpC enzymes. No carbapenemase-producing strains were detected. Pulsed-field gel electrophoresis showed that 21 E. coli strains (91%) were genetically unrelated isolates. Increasing resistance of bacteria to antibiotic agents poses a serious issue for both human and veterinary medicine globally. For humans, a potential source of resistant bacteria may be animals or their products entering the human food chain, for example poultry. The presented study extends existing knowledge about the occurrence of resistant bacteria in poultry in Moravia and describes the phenotype and genotype of their resistance to antibiotics.