Objectives: The objective of this study was to determine the activity of fidaxomicin and comparator antimicrobials against Clostridium difficile isolated from patients with C. difficile infection (CDI) in Australian hospitals and in the community. Methods: One private and one public laboratory from five states in Australia submitted a total of 474 isolates/PCR-positive stool samples during three collection periods in August-September 2013 (n=175), February-March 2014 (n=134) and August-September 2014 (n=165). Isolate identification was confirmed by selective culture for C. difficile and a proportion of isolates from each state were characterized by PCR for toxin genes and PCR ribotyping. MICs of fidaxomicin and eight comparator antimicrobials were determined for all isolates using agar methodology. Results: Site collection yielded 440 isolates of C. difficile and PCR revealed a heterogeneous strain population comprising 37 different PCR ribotypes (RTs), 95 of whichwere positive for tcdA and tcdB (A+B+). The most common RTs were 014 (29.8 ) and 002 (15.9 ). Epidemic RT 027 was not identified; however, small numbers of virulent RTs 078 and 244 were found. Resistance to vancomycin, metronidazole and fidaxomicin was not detected and resistance to moxifloxacin was very low (3.4 ). Fidaxomicin showed potent in vitro activity against all 440 isolates (MIC50/MIC90 0.03/0.12 mg/L) and was superior to metronidazole (MIC50/MIC90 0.25/0.5 mg/L) and vancomycin (MIC50/MIC90 1/2 mg/L). Conclusions: These data confirm the potent in vitro activity of fidaxomicin against C. difficile. Moreover, this study provides an important baseline for ongoing long-term surveillance of antimicrobial resistance and prospective tracking of prominent and emerging strain types. ? The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.