Abstract The aim of this study was to investigate the molecular basis for observed high-level quinolone and macrolide resistance in poultry Campylobacter isolates. Seventeen Campylobacter isolates displaying high-level resistance to nalidixic acid, ciprofloxacin and/or erythromycin were investigated. Minimum inhibitory concentrations were initially determined using both the broth microdilution and E-test methods. The contribution of target gene mutations and active efflux to the observed resistances were then investigated using PCR and sequencing methods. High-level resistance to nalidixic acid was attributed to amino acid substitutions Thr-86-Ile and Asn-203-Ser in GyrA in some but not all isolates. Contrary to previous reports, the Thr-86-Ile substitution did not confer universal resistance to all quinolones. Strains displaying a high level of resistance to erythromycin carried the 23S rRNA transition mutation A2075G and/or carried mutations in the L4 and/or L22 ribosomal-encoding proteins. Interestingly and in contrast to previous studies, not all of the isolates carrying substitutions within the β-hairpin region of the L22 ribosomal protein displayed erythromycin resistance. With the exception of a single isolate, efflux did not contribute to either quinolone or macrolide resistance. This study further expands our understanding of the molecular basis of quinolone and macrolide resistance in Campylobacter spp. and suggests that other factors, remaining to be elucidated, may also contribute to the resistant phenotypes observed.