ABSTRACTControl of hospital-associatedEnterococcus faeciuminfection is a strenuous task due to the difficulty of identifying transmission routes and the persistence of this nosocomial pathogen despite the implementation of infection control measures that have been successful with other important nosocomial pathogens. This study provides a comprehensive analysis of over 100E. faeciumisolates collected from 66 cancer patients at the University of Arkansas for Medical Sciences (UAMS) between June 2018 and May 2019. In the top-down approach used in this study, we employed, in addition to the 106E. faeciumUAMS isolates, a filtered set of 2,167E. faeciumstrains from the GenBank database to assess the current population structure ofE. faeciumspecies and, consequently, to identify the lineages associated with our clinical isolates. We then evaluated the antibiotic resistance and virulence profiles of hospital-associated strains from the species pool, focusing on antibiotics of last resort, to establish an updated classification of high-risk and multidrug-resistant nosocomial clones. Further investigation of the clinical isolates collected from UAMS patients using whole-genome sequencing analytical methodologies (core genome multilocus sequence typing [cgMLST], core single nucleotide polymorphism [coreSNP] analysis, and phylogenomics), with the addition of patient epidemiological data, revealed a polyclonal outbreak of three sequence types occurring simultaneously in different patient wards. The integration of genomic and epidemiological data collected from the patients increased our understanding of the relationships and transmission dynamics of theE. faeciumisolates. Our study provides new insights into genomic surveillance ofE. faeciumto assist in monitoring and further limiting the spread of multidrug-resistantE. faecium.IMPORTANCEEnterococcus faeciumis a member of the gastrointestinal microbiota. Although its virulence is low in healthy, immunocompetent individuals,E. faeciumhas become the third leading cause of health care-associated infections in the United States. This study provides a comprehensive analysis of over 100E. faeciumisolates collected from cancer patients at the University of Arkansas for Medical Sciences (UAMS). We employed a top-down analytical approach (from population genomics to molecular biology) to classify our clinical isolates into their genetic lineages and thoroughly evaluate their antibiotic resistance and virulence profiles. The addition of patient epidemiological data to the whole-genome sequencing analytical methodologies performed in the study allowed us to increase our understanding of the relationships and transmission dynamics of theE. faeciumisolates. This study provides new insights into genomic surveillance ofE. faeciumto help monitor and further limit the spread of multidrug-resistantE. faecium.