Objective To describe the current status of healthcare-associated infection (HAI) in medical institutions (MIs) in Guizhou Province. Methods A cross-sectional survey was conducted to investigate the prevalence rates of HAI in 186 MIs in Guizhou Province from July 15 to December 31, 2022. Results A total of 89 409 hospitalized patients were surveyed, with 1 066 cases of HAI. The prevalence rate of HAI was 1.19%, with Guiyang City having the highest HAI rate (1.38%) and Bijie City having the lowest (0.77%). Among HAI sites, the proportion of lower respiratory tract was the highest (38.96%), followed by urinary tract (14.42%) and skin soft tissue (8.00%). The department with the highest prevalence rate of HAI was general intensive care unit (11.39%), followed by department of hematology (6.68%) and department of neurosurgery (4.83%). The main pathogens of HAI was Gram-negative bacteria, accounting for 66.79%. The usage rate of antimicrobial agents was 36.06%. Among patients underwent class Ⅰ incision surgery, the prevalence rate of HAI was 3.30%, and the usage rate of antimicrobial agents was 20.87%. There were statistically significant differences in the usage rate of antimicrobial agents, the HAI prevalence rate of patients who underwent class Ⅰ incision surgery, and the preventive usage rate of antimicrobial agents in patients in MIs with different bed numbers(χ²=250.218, 16.454, 99.891, respectively all P < 0.01). Conclusion It is necessary to strengthen the management of infection prevention and control in hospitals in Guiyang and Bijie Cities, as well as target monitoring and infection control on surgical site infection of class Ⅰ incision surgery in general ICUs and large-scale MIs.