Objective To study the intervention effect of nursing staff’s participation in antimicrobial stewardship (AMS) on the rational use of antimicrobial agents, and explore its role in constructing a scientific healthcare-associated infection (HAI) control management. Methods The data on perioperative prophylactic use of antimicrobial agents, surgical-related HAI control, and pathogen detection before therapeutic use of antimicrobial agents among hospitalized patients in a hospital from January 2016 to December 2024 were collected. Relevant evaluation indicators before and after nursing staff participating in AMS were compared. 2016-2018, 2019-2021, and 2022-2024 were stages before intervention, during intervention, and after intervention, respectively. Results After nursing staff participated in AMS, the use rate of prophylactic antimicrobial agents 0.5-1 hour before surgery and discontinuation rate of antimicrobial agents within 24 hours after class I incision surgery increased from 64.54% and 81.41% before intervention to 75.31% and 84.56% after intervention, respectively. Incidences of surgical-related HAI and surgical site infection in patients decreased from 3.11% and 0.96% before intervention to 1.37% and 0.17% after intervention, respectively. Pathogen detection rates before restricted- and special-grade antimicrobial agents treatment increased from 50.80% and 68.70% before intervention to 55.19% and 80.53% after intervention, respectively. Proportion of blood specimen from which coagulase-negative Staphylococcus was detected decreased from 29.30% before intervention to 21.26% after intervention. Proportion of respiratory specimen from which Haemophilus influenzae was detected increased from 2.00% to 3.98%. Differences were all statistically significant (all P<0.05). Conclusion As important members of the AMS team, nursing staff can effectively reduce irrational antimicrobial use, optimize medication timing and duration, and have a positive effect on ensuring patient safety through participating in the use and management of antimicrobial agents in hospitalized patients.