Background: Consistent compliance with evidence- based guidelines is challenging yet critical to patient safety. We conducted a qualitative study to explore the underlying causes for non-compliance with evidence- based guidelines aimed at preventing four types of healthcare-associated infections in the surgical intensive care unit (SICU) setting. Methods: Twenty semistructured interviews were conducted with attending physicians (3), residents (2), nurses (6), quality improvement coordinators (3), infection control practitioners (2), respiratory therapists (2) and pharmacists (2) in two SICUs. Using a grounded theory approach, we performed thematic analyses of the interviews. Results: The concept of systems ambiguity to explain noncompliance with evidence-based guidelines emerged from the data. Ambiguities hindering consistent compliance were related to tasks, responsibilities, methods, expectations and exceptions. Strategies reported to reduce ambiguity included clarification of expectations from care providers with respect to guideline compliance through education, use of visual cues to indicate the status of patients with respect to a particular guideline, development of tools that provide an overview of information critical for guideline compliance, use of standardised orders, clarification of roles of care providers and use of decision-support tools. Conclusions: The concept of systems ambiguity is useful to understand causes of non-compliance with evidence- based guidelines aimed at reducing healthcare-associated infections. Multi-faceted interventions are needed to reduce different ambiguity types, hence to improve guideline compliance.