Background: Sustained injudicious and indiscriminate use of antimicrobials has exerted selection pressure for developing antimicrobial resistance (AMR), requiring behaviour change from healthcare professionals (HCPs) based on their knowledge, attitudes and practices (KAP) on antimicrobials, AMR and antimicrobial stewardship (AMS). Methods:A cross-sectional online questionnaire-based survey was conducted nationally amongst doctors, pharmacists and nurses from November 2017 to January 2018. The questionnaire comprised demographic information and KAP questions.Results: Respondents comprised of 1120 doctors, 744 pharmacists and 659 nurses. Antimicrobial resistance was considered a severe problem globally and nationally by majority of HCPs. Self-assessment of knowledge revealed gaps in understanding of antimicrobials, AMR and AMS. Confidence scores in prescribing by doctors, pharmacists and nurses were 57.82%, 32.88% and 45.28%, respectively. Doctors, 441 (45.2%) indicated no confidence in using combination therapy. Prescribing correctly showed a confidence level of 33.99% from 436 doctors, 41.88% from nine pharmacists and 35.23% from 107 nurses. Healthcare professionals ( 1600[91.22%]) stated educational campaigns would combat AMR. Only 842 (40.13%) HCPs attended training on these topics and 1712 (81.60%) requesting more education and training. Conclusion:This is the first comparative survey on KAP of practising doctors, pharmacists and nurses in South Africa. Doctors had the highest knowledge score followed by nurses and pharmacists. Practice scores did not corroborate knowledge and the higher attitude scores. Gaps in KAP were evident. Healthcare professionals indicated the need for more education and training, thus requiring a review of pre-service and in-service education and training in addition to continued professional development programmes for practising HCPs.