Objective To understand the current situation of healthcare-associated infection (HAI) in China, provide data support and decision-making basis for formulating scientific and effective strategies for HAI prevention and control. Methods A nationwide cross-sectional survey on HAI was conducted among various types and levels of medical institutions in China according to a unified protocol of bedside surveys and case investigations. Results In 2024, a total of 5 736 medical institutions and 2 751 765 patients were surveyed. Among them, 34 889 HAI cases were identified, with a prevalence rate of 1.27%. The number of HAI episodes was 38 032, and case prevalence rate was 1.38%. The prevalence rate of HAI in medical institutions in different regions of China ranged from 0.66% to 2.35%. Among medical institutions of different scales, those with a bed capacity of ≥900 had the highest incidence of HAI, reaching 1.65%. The most common infection site was the lower respiratory tract (44.66%), followed by the urinary tract (12.94%), surgical site (9.32%), upper respiratory tract (7.02%), and bloodstream infection (5.78%). The top 3 departments with the highest HAI rates were the general intensive care unit (10.02%), department of neurosurgery (5.51%), and department (group) of hematology (5.34%). A total of 23 238 strains of HAI pathogens were detected, with 10 714 strains (46.10%) from lower respiratory tract specimens. The top 5 detected strains were Klebsiella pneumoniae (14.76%), Pseudomonas aeruginosa (13.33%), Escherichia coli (12.79%), Acinetobacter baumannii (9.23%), and Staphylococcus aureus (7.88%). 231 944 patients underwent class I incision surgery were monitored, with 1 647 cases experienced surgical site infection, and the prevalence rate of surgical site infection was 0.71%. The number of patients who should undergo pathogen detection (patients receiving therapeutic and therapeutic combined prophylactic antimicrobial agents) was 715 179, while the actual number was 480 492, with a pathogen detection rate of 67.18%. 425 225 patients received pathogenic detection before treatment, with a detection rate of 59.46%. Conclusion The overall HAI prevalence in China is lower, showing disparities among medical institutions of different regions and scales. Therefore, precise implementation of measures is necessary for HAI prevention and control, with a focus on high-risk institutions and high-risk departments, key areas, and critical procedures. All levels of medical institutions should continuously reduce the incidence of HAI by strengthening monitoring, standardizing the use of antimicrobial agents, and reinforcing basic HAI prevention and control measures.