The aim of this study was to assess the possible circulation of genetic resistance determinants and chromosomal point mutations in quinolone-resistant Escherichia coli isolated from livestock from central Italy. Forty-nine E. coli isolates were recovered from animals during the surveillance activities of the Istituto Zooprofilattico Abruzzo e Molise (IZSA&M), Italy, over 2 years. The plasmid resistance determinants and point mutations in DNA gyrase and topoisomerase IV were characterized by PCR and DNA sequencing. Of the 49 E. coli isolates, 34 were resistant to nalidixic acid, 4 to ciprofloxacin and 11 to nalidixic acid, ciprofloxacin and enrofloxacin. Chromosomal point mutations were found in gyrA gene (Ser83Leu and Asp87Asn) and gyrB (Gln434His, Lys444Arg and Gly435Val). We also report the simultaneous presence of qnrS1 quinolone resistance determinant, dfrA1-aadA22 gene cassettes and amino acid substitution Ser83Leu in the gyrA gene in an E. coli strain resistant only to nalidixic acid.