With regard to antibiotic resistance studies in various model animals in the urban environment, the presented study focused on the rook, many behavioural and ecological aspects of which are important from an epidemiological point of view. A total of 130 Escherichia coli strains isolated from rook faeces during a two-year period (2011–2012) were investigated for antibiotic resistance and virulence. Resistance to ampicillin (60%) and streptomycin (40%) were the most frequent, followed by resistance to fluoroquinolones (ciprofloxacin-22% and enrofloxacin-24%), tetracycline (18%), cotrimoxazol (17%) and florfenicol (14%). Ceftiofur resistance occured in 10.7% of strains and cefquinom resistance in 1.5% of strains. Twenty-five E.coli strains with a higher level of MICs of cephalosporins (over 2mg/L of ceftazidime and ceftriaxon) and fluoroquinolones were selected for detection of betalactamase genes (CTX-M, CMY), plasmid-mediated quinolone resistance qnrS, integrase 1, and for APEC (avian pathogenic E.coli) virulence factors (iutA, cvaC, iss, tsh, ibeA, papC, kpsII). Genes of CTX-M1, CMY-2, integrase 1, papC, cvaC, iutA were detected in one strain of E.coli, and qnrS, integrase 1, iss, cvaC, tsh were detected in another E.coli. DNA microarray revealed the absence of verotoxin and enterotoxin genes and pathogenicity islands. The results show that rooks can serve as a reservoir of antibiotic-resistant E. coli with avian pathogenic virulence factors for the human population, and potentially transmit such E.coli over long distances.