From 1 January to 31 December 2021, forty-eight institutions around Australia participated in the Australian Enterococcal Surveillance Outcome Programme (AESOP). The aim of AESOP 2021 was to determine the proportion of enterococcal bacteraemia isolates in Australia that were antimicrobial resistant, and to characterise the molecular epidemiology of the Enterococcus faecium isolates. Of the 1,297 unique episodes of enterococcal bacteraemia investigated, 94.4% were caused by either E. faecalis (54.1%) or E. faecium (40.3%). Ampicillin resistance was detected in one E. faecalis isolate and in 89.3% of E. faecium isolates. Vancomycin non-susceptibility was not detected in E. faecalis but was detected in 37.9% of E. faecium. Overall, 39.6% of E. faecium harboured the vanA and/or vanB genes. For the vanA/vanB positive E. faecium isolates, 35.8% harboured the vanA gene and 64.2% the vanB gene. Although the percentage of vancomycin-resistant E. faecium bacteraemia isolates was significantly lower than that reported in the 2020 AESOP report (presumably due to the COVID-19 elective surgery restrictions placed on hospitals), it remains substantially higher than that recorded in most European countries. Isolates of E. faecium consisted of 73 multi-locus sequence types (STs); 77.2% of isolates were classified into seven major STs each containing more than ten isolates. All major STs belonged to clonal cluster (CC) 17, a major hospital-adapted polyclonal E. faecium cluster. The major STs (ST17, ST1424, ST796, ST78, ST80, ST1421 and ST555) were found across most regions of Australia. The predominant ST was ST17 which was identified in all regions except the Northern Territory. Overall, 46.5% of isolates belonging to the seven major STs harboured the vanA or vanB gene. The AESOP 2021 has shown that enterococcal bacteraemia episodes in Australia are frequently caused by polyclonal ampicillin-resistant high-level gentamicin resistant vanA- or vanB-positive E. faecium which have limited treatment options.