OBJECTIVE To assess resource allocation and costs associated with US hospitals preparing for the possible spread of the 2014-2015 Ebola virus disease (EVD) epidemic in the United States. METHODS A survey was sent to a stratified national probability sample (n=750) of US general medical/surgical hospitals selected from the American Hospital Association (AHA) list of hospitals. The survey was also sent to all children's general hospitals listed by the AHA (n=60). The survey assessed EVD preparation supply costs and overtime staff hours. The average national wage was multiplied by labor hours to calculate overtime labor costs. Additional information collected included challenges, benefits, and perceived value of EVD preparedness activities. RESULTS The average amount spent by hospitals on combined supply and overtime labor costs was $80,461 (n=133; 95% confidence interval [CI], $56,502-$104,419). Multivariate analysis indicated that small hospitals (mean, $76,167) spent more on staff overtime costs per 100 beds than large hospitals (mean, $15,737; P<.0001). The overall cost for acute-care hospitals in the United States to prepare for possible EVD cases was estimated to be $361,108,968. The leading challenge was difficulty obtaining supplies from vendors due to shortages (83%; 95% CI, 78%-88%) and the greatest benefit was improved knowledge about personal protective equipment (89%; 95% CI, 85%-93%). CONCLUSIONS The financial impact of EVD preparedness activities was substantial. Overtime cost in smaller hospitals was >3 times that in larger hospitals. Planning for emerging infectious disease identification, triage, and management should be conducted at regional and national levels in the United States to facilitate efficient and appropriate allocation of resources in acute-care facilities. Infect Control Hosp Epidemiol 2017;38:405-410.