Objectives: Explore perceptions and attitudes of nurses in regard to antimicrobial stewardship (AMS), their roles as nurses, and identify differences in perceptions and attitudes across paediatric and adult settings. Methods: Electronic survey administered to nursing staff across three public Australian tertiary institutions with AMS facilitated by a shared electronic approval and decision support system. Results: Overall 65% (93/142) of nurses who completed the survey were familiar with the term AMS, and 75% recognised that they were expected to have a role alongside other disciplines, including ward pharmacists (paediatric 88%, adult 73%; p = 0.03). Hand hygiene and infection control (86%), patient advocacy (85%) and knowledge of antimicrobials (84%) were identified most often as AMS roles for nurses. However, 57% of nurses reported that their knowledge of antimicrobials was minimal or limited. Nurses generally agreed that requirement to obtain approval is an effective way to reduce inappropriate antimicrobial use (median scores: paediatric 2.0 [agree], adult 1.0 [strongly agree]; p = 0.001). Only 35% of paediatric and 58% of adult nurses perceived that their role includes ensuring approval for restricted antimicrobials (p < 0.01). Most nurses identified AMS teams (85%), pharmacists (83%) and infection control teams (paediatric 68%, adult 84%; p = 0.04) as sources of AMS support. Areas of interest for support and education included appropriate antimicrobial selection (73%) and intravenous to oral antimicrobial switch (paediatric 65%, adult 81%, p = 0.03). Conclusion: Nurses consider AMS activities within their roles, but are underutilised in AMS programs. Further engagement, education, support and acknowledgement are required to improve nursing participation.