Healthcare-associated infection (HAI) is a major cause of morbidity, mortality and cost, which vary widely by region and hospital. In this case-control study, we calculated losses attributable to HAI in central China. A total of 2976 patients in 10 hospitals were enrolled, and the incidence rate of HAI (range, 0.88–4.15%) was significantly, but negatively associated with the cost per 1000 beds of its prevention (range, $24 929.76–$53 146.41; r = −0.76). The per capita economic loss attributable to HAIs was $2047.07 (interquartile range, $327.63–$6429.17), mainly from the pharmaceutical cost (median, $1044.39). The HAIs, which occurred in patients with commercial medical insurance, affected the haematologic system and caused by Acinetobacter baumannii, contributed most to the losses (median, $3881.55, $4734.20 and $9882.75, respectively). Furthermore, the economic losses attributable to device-associated infections and hospital-acquired multi-drug resistant bacteria were two to four times those of the controls. The burden attributable to HAI is heavy, and opportunities for easing this burden exist in several areas, including that strengthening antibiotic stewardship and practicing effective bundle of HAI prevention for patients carrying high-risk factors, for example, elders or those with catheterisations in healthcare institutions, and accelerating the medical insurance payment system reform based on diagnosis-related groups by policy-making departments.