Background Surgical antimicrobial prophylaxis (SAP) is a leading indication for antibiotic use in the hospital setting, with demonstrated high rates of inappropriateness. Decision-making for SAP is complex and multifactorial. A greater understanding of these factors is needed to inform the design of targeted antimicrobial stewardship interventions and strategies to support the optimization of SAP and its impacts on patient care. Methods A qualitative case study exploring the phenomenon of SAP decision-making. Focus groups were conducted with surgeons, anaesthetists, theatre nurses and pharmacists across one private and two public hospitals in Australia. Thematic analysis was guided by the Theoretical Domains Framework (TDF) and the Capabilities, Opportunities, Motivators-Behaviour (COM-B) model. Results Fourteen focus groups and one paired interview were completed. Ten of the fourteen TDF domains were identified as relevant. Thematic analysis revealed six significant themes mapped to the COM-B model, and subthemes mapped to the relevant TDF domains in a combined framework. Key themes identified were: 1) Low priority for surgical antimicrobial prophylaxis prescribing skills; 2) Prescriber autonomy takes precedence over guideline compliance; 3) Social codes of prescribing reinforce established practices; 4) Need for improved communication, documentation and collection of data for action; 5) Fears and perceptions of risk hinder appropriate SAP prescribing; and 6) Lack of clarity regarding roles and accountability. Conclusions SAP prescribing is a complex process that involves multiple professions across the pre-, intra- and post-operative surgical settings. The utilisation of behaviour change frameworks to identify barriers and enablers to optimal SAP prescribing supports future development of theory-informed antimicrobial stewardship interventions. Interventions should aim to increase surgeon engagement, enhance the prioritisation of and accountability for SAP, and address the underlying social factors involved in SAP decision-making, such as professional hierarchy and varied perceptions or risks and fears.