Objective To understand the current situation of healthcare-associated infection (HAI) among hospitalized patients in Yunnan area from 2020 to 2022, and provide reference for optimizing corresponding prevention and control measures. Methods Data of HAI prevalence rate survey (cross-sectional survey) in Yunnan area from 2020 to 2022 were collected through Yunnan Provincial HAI Quality Control Center Surveillance System, and su-mmarized. Statistical analysis was performed with SPSS 25.0 software. Results From 2020 to 2022, 117 695, 109 691, and 137 188 patients were surveyed respectively, and the prevalence rates of HAI were 1.58%, 1.47%, and 1.29%, respectively, with statistically significant difference (χ²=36.853, P<0.001). Lower respiratory tract (n=2 526, 43.60%) and urinary tract (n=785, 13.55%) were the major infection sites. General intensive care unit (ICU) had the highest HAI prevalence rate (10.95%-16.47%) in all three prevalence surveys. The main pathogens causing HAI were Gram-negative bacteria, with Escherichia coli being the first, followed by Klebsiella pneumoniae, while the major Gram-positive bacteria was Staphylococcus aureus. Antimicrobial use rates in patients were 30.96%-33.91%, showing an upward trend (χ²＝257.586, P＜0.001). The main purpose of antimicrobial use was for treatment (accounting for 76.46%-78.62%). Antimicrobial use was mainly single use, with a slight increase in combined use of three antimicrobials and above, increasing from 0.66% to 0.98%. The pathogenic detection rate for therapeutic antimicrobial use showed an upward trend (χ²＝94.823, P＜0.001). Surgical site infection rates in patients undergoing class Ⅰ incision surgery were 0.77%-1.22%; the prophylactic antimicrobial use rates in patients undergoing class Ⅰ incision surgery in three years were 19.40%, 19.11%, and 20.93%, respectively, both with statistically significant difference (χ²=94.823, 10.179, both P=0.006). Conclusion The prevalence rate of HAI among hospitalized patients in Yunnan area has decreased, however, the prevention and control of HAI pathogens, as well as the rational use of prophylactic antimicrobial agents for class Ⅰ incision surgical patients are difficult, and represent the focus of future work.