Background State-specific, health care-associated infection (HAI) cost estimates have not been calculated to guide Department of Public Health efforts and investments. Methods We completed a cost identification study by conducting a survey of 117 acute care hospitals in NC to collect surveillance data on patient-days, device-days, and surgical procedures during 1 year. We then calculated expected rates and direct hospital costs of surgical site infections (SSI), Clostridium difficile infection, and 3 selected device-related HAIs for hospitals and the entire state using reference data sets such as the National Healthcare Safety Network. Results In total, 67 (53%) hospitals responded to the survey. The median bed size of respondent hospitals was 140 (interquartile range, 66-350). A “standard” NC hospital diagnosed approximately 100 HAI each year with estimated costs of $985,000 to $2.7 million. The most common HAI was SSI (73%). Costs related to SSI accounted for 87% to 91% of overall costs. In total, the overall direct annual cost of these 5 selected HAIs was estimated to be between $124.1 and $347.8 million in 2009 for the state of NC. Conclusion Using conservative estimates, HAI led to costs of more than $100 million in acute care hospitals in the state of NC in 2009. The majority of costs were due to SSI.