<p>Health-care-associated infections (HAIs) are a global threat. Microbial whole-genome sequencing (WGS) can strengthen HAI prevention strategies by enabling high-resolution detection and tracking of pathogen transmission and predicting clinically relevant phenotypic traits, such as antibiotic resistance and virulence. Although WGS continues to serve as a vital adjunct in outbreak responses, its role has evolved to include prospective pathogen surveillance and epidemiological monitoring. In this Review, we provide an overview of the epidemiology and microbiology of HAIs and highlight actionable insights gained by inclusion of WGS in HAI investigation and surveillance, with an emphasis on high-priority antibiotic-resistant bacterial pathogens. We explore the value of incorporating plasmid analysis into investigations and emphasize the importance of integrating genomic data with clinical and epidemiological metadata to support accurate transmission inferences. Critical methodological decisions are examined, including strategies for sample selection and the determination of an appropriate single nucleotide variant threshold to identify patients linked by transmission. The need for capacity building in low-and-middle-income countries, which bear a disproportionate burden of HAIs, is discussed. Together, these considerations underscore the transformative potential of WGS to inform targeted, data-driven interventions and advance global efforts to reduce the burden of HAIs.</p>