Controlling the use of the most critically important antimicrobials (CIAs) in food animals has been identified as one of the key measures required to curb the transmission of antimicrobial resistant bacteria from animals to humans. Expanding the evidence demonstrating the effectiveness of restricting CIA usage for preventing the emergence of resistance to key drugs amongst commensal organisms in animal production would do much to strengthen international efforts to control antimicrobial resistance (AMR). As Australia has strict controls on antimicrobial use in layer hens, and internationally comparatively low levels of poultry disease due to strict national biosecurity measures, we investigated whether these circumstances have resulted in curtailing development of critical forms of AMR. The work comprised a cross-sectional national survey of 62 commercial layer farms with each assessed for AMR inEscherichia coliisolates recovered from faeces. Minimum inhibitory concentration analysis using a panel of 13 antimicrobials was performed on 296 isolates, with those exhibiting phenotypic resistance to fluoroquinolones (a CIA) or multi-class drug resistance (MCR) subjected to whole genome sequencing. Overall, 53.0% of isolates were susceptible to all antimicrobials tested, and all isolates were susceptible to cefoxitin, ceftiofur, ceftriaxone, chloramphenicol and colistin. Resistance was observed for amoxicillin-clavulanate (9.1%), ampicillin (16.2%), ciprofloxacin (2.7%), florfenicol (2.4%), gentamicin (1.0%), streptomycin (4.7%), tetracycline (37.8%) and trimethoprim/sulfamethoxazole (9.5%). MCR was observed in 21 isolates (7.0%), with two isolates exhibiting resistance to four antimicrobial classes. Whole genome sequencing revealed that ciprofloxacin-resistant (fluoroquinolone) isolates were devoid of both known chromosomal mutations in the quinolone resistance determinant regions and plasmid-mediated quinolone resistance genes (qnr)—other than in one isolate (ST155) which carried theqnrSgene. Two MCRE.coliisolates with ciprofloxacin-resistance were found to be carrying known resistance genes includingaadA1,dfrA1,strA,strB,sul1,sul2,tet(A),blaTEM-1B,qnrS1andtet(A). Overall, this study found thatE.colifrom layer hens in Australia have low rates of AMR, likely due to strict control on antimicrobial usage achieved by the sum of regulation and voluntary measures.