OBJECTIVE: To determine whether the gender of clinicians making antimicrobial stewardship recommendations has an impact on intervention acceptance rate. DESIGN: A retrospective, multivariable analysis of antimicrobial stewardship prospective audit and feedback outcomes. SETTING: A multisite healthcare system including Mayo Clinic Rochester (MN), Mayo Clinic Arizona, Mayo Clinic Florida and 17 health-system hospital sites, where prospective audit and feedback is performed and documented within an electronic tool embedded in the medical record. PARTICIPANTS: The study included 143 Mayo Clinic clinicians (84 cisfemales and 59 cismales). METHODS: Outcomes were analyzed from July 1, 2017, to June 30, 2022, for intervention rates, communication methods, and intervention acceptance by clinician gender, profession, patient age, and intensive care unit (ICU) status of patient. RESULTS: Of 81,927 rules, 71,729 rules met study inclusion. There were 18,175 (25%) rules associated with an intervention. Most of the rules were reviewed by pharmacists (86.2%) and stewardship staff (85.5%). Of 10,363 interventions with an outcome documented, 8,829 (85.2%) were accepted and 1,534 (14.8%) were rejected. Female clinicians had 6,782 (86.5%) of 7,843 interventions accepted, and male clinicians had 2,047 (81.2%) of 2,520 interventions accepted (P = .19). Female patients had more interventions than male patients (female vs male: 25.9% vs 24.9%; OR, 1.04; 95% CI, 1.02-1.08; P = .001). Patients in the ICU had a significantly lower intervention acceptance rate (ICU vs non-ICU: 78.2% vs 86.7%; OR, 0.56; 95% CI, 0.45-0.7; P < .001). CONCLUSIONS: Female and male clinicians were equally effective at prospective audit and feedback in a multisite antimicrobial stewardship program. Patients in the ICU were less likely to have stewardship interventions accepted.