Antimicrobial stewardship programmes (ASPs) facilitate appropriate antimicrobial use and require contextualization for optimal functioning. We aimed to investigate perceptions of and antimicrobial resistance (AMR) and ASPs among healthcare workers in academic and nonacademic hospitals. Cross-sectional survey. Three academic (Charlotte Maxeke Johannesburg Academic, Inkosi Albert Luthuli, Tygerberg) and three nonacademic hospitals (Leratong, Prince Mshiyeni Memorial, and Paarl) in South Africa from January to June 2022. Doctors, nurses, and pharmacists. Voluntary questionnaire using Google Forms, encompassing AMR, ASPs, and selected discipline-specific components. Participants comprised 79 doctors (50 academic), 178 nurses (169 academic), and 21 pharmacists (18 academic) and were female predominant. AMR was a problem in academic hospitals (74.7% vs 51.2%, p 0.004); 73.5% overall reported inappropriate antimicrobial use as a major contributor. Adequate education on antimicrobials occurred in only 36.4% overall. Microbiological testing guided therapy more often in nonacademic settings (80.0% vs 50.2%, p <0.001). In both settings, antimicrobial availability drove selection in 48.2%. Overall, ASPs improved patient care (89.8%) and reduced antimicrobial use (86.9%), although felt to override prescriber autonomy in academic settings (29.4% vs 7.5%, p 0.007), mainly among nurses. Only 50.2% reported successful local ASPs. A minority of pharmacists (20.0%) reported sufficient hospital support for ASPs. Education, involvement of infection control staff, and inclusion of nurses in ASPs were most impactful on AMR. Selected healthcare worker perspectives differ by category and setting and can be targeted to improve ASPs. Further studies should target a higher number of clinical staff in both settings.