Objective To evaluate the impact of healthcare-associated infection (HAI) control culture construction on the hand hygiene (HH) compliance of health care workers (HCWs), and provide a basis for strengthening HAI management. Methods HCWs in a hospital in Xi’an City were selected as the research objects. On the occasion of World HH Day, a series of publicity activities on HH as well as HAI prevention and control were held. Pre-activities period was April 1-30, 2024. During May 1-31, 2024, a series of publicity activities on HH as well as HAI prevention and control were carried out. The post-activities investigation period was June 1-30, 2024. During December 1-31, 2024, HH compliance survey was carried out in batches in the whole hospital (including key departments). HH compliance at different stages was compared. Results After a series of publicity activities on HH as well as HAI prevention and control, HCWs’ HH compliance rate was improved. HH compliance rate of nursing staff increased from 70.15% to 85.11%; HH compliance rate of HCWs before contacting with patients increased from 47.83% to 78.38%; both with statistically significant difference (both P<0.05). During December 1-31, 2024, a batch survey on HH compliance of the whole hospital (including key departments) showed that the HH compliance rates of medical and nursing staff in key departments were higher than those of the whole hospital, and the differences were statistically significant (all P<0.05). HH compliance rates before aseptic operation and after contacting with patients in key departments were both higher than those of the whole hospital, and the differences were statistically significant (all P<0.05). Conclusion Publicity activities on HH as well as HAI prevention control can improve HH compliance rate of HCWs. HAI managers should pay more attention to the general departments and improve HH compliance rate of HCWs.