Listeria monocytogenes is of public health concern as it is a food borne pathogen responsible for some of the highest hospitalisation and mortality rates worldwide. Elderly, the immunocompromised, pregnant women and the foetus are the populations most at risk of developing invasive listeriosis. Listeriosis patients rely on the ability of antibiotics to destroy L. monocytogenes however, with the expansion of antibiotic resistance in other bacterial species, similar trends in L. monocytogenes poses a high risk to these patients. This study characterised 100 Australian L. monocytogenes isolates from food production chains with: serotyping, antisera and multiplex-PCR; Pulsed-Field Gel Electrophoresis; Whole Genome Sequencing and Multi-Locus Sequence Typing. The susceptibility of these isolates against 4 antibiotics, comprising ciprofloxacin, erythromycin, penicillin and tetracycline was assessed using M.I.C.Evaluator™ strips. Finally, comparative genomic analysis was performed to determine the presence of genes responsible for antibiotic resistance (tetL, tetM, tetK, ermA, ermB, ermC, dfrD, dfrG, vanA and vanB). Serological analysis identified that serotype 1/2a (50%) was the predominant serotype followed by 1/2b (29%), 4b (7%), 1/2c (4%), 3a (3%), 4d (3%), 3b (2%), 4a and 4e (both 1%). All isolates were susceptible to penicillin and tetracycline, however two serotype 4b isolates were identified as being ciprofloxacin-resistant, one of which was also resistant to erythromycin. The cause of the ciprofloxacin-resistance in isolates 2948 and Lm16-001 may be the result of two unique mutations identified in the fluoroquinolone efflux pump repressor gene, fepR at different nucleotide locations. The results from this study indicate a low rate of antibiotic resistance to four antibiotics in L. monocytogenes isolates from Australian food production environments. There is limited knowledge on the potential role of these mutations in ciprofloxacin-resistance and further study is required. Additionally, the significance of this ciprofloxacin-resistance is unknown due to a lack of L. monocytogenes antibiotic resistance data in Australia. The increasing reports of antibiotic resistance in L. monocytogenes globally suggests further surveillance of this important human pathogen is required.