Abstract: The detection of pathogens with decreased susceptibility to antibacterial and elucidating the molecular mechanism of resistance are of epidemiological and clinical interest in animal husbandry. The 21 Haemophilus parasuis clinical isolates and one American Type Culture Collection reference strains of Actinobacillus pleuropneumoniae (ATCC 27090) were screened for susceptibility to nalidixic acid and enrofloxacin by the microdilution methods. The nalidixic acid MICs for all of the isolates were ≥2 μg mL-1 and the enrofloxacin MICs for isolates were ≤8 μg mL-1. In addition, the Quinolone Resistance-Determining Regions (QRDRs) of gyrA and parC of all strains were sequenced. Strains for which nalidixic acid MICs were ≤4.0 μg mL-1 lacked modifications in the QRDRs of GyrA and ParC. In contrast, all strains for which nalidixic acid MICs were ≥8 μg mL-1 exhibited one or more amino acid changes in GyrA and ParC. Mutations in GyrA is the major resistance mechanism of Haemophilus parasuis to nalidixic acid and enrofloxacin.