INTRODUCTION: Important culture-change initiatives (e.g. improving hand hygiene compliance) are frequently associated with variable uptake among different healthcare worker (HCW) categories. Inherent personality differences between these groups may explain change uptake and help improve future intervention design. MATERIALS AND METHODS: We used an innovative personality-profiling tool (ColourGrid®) to assess personality differences among standard HCW categories at five large Australian hospitals using two data sources (HCW participant surveys [PS] and generic institution-wide human resource [HR] data) to: a) compare the relative accuracy of these two sources; b) identify differences between HCW groups and c) use the observed profiles to guide design strategies to improve uptake of three clinically-important initiatives (improved hand hygiene, antimicrobial stewardship and isolation procedure adherence). RESULTS: Results from 34,243 HCWs (HR data) and 1045 survey participants (PS data) suggest that HCWs were different from the general population, displaying more individualism, lower power distance, less uncertainty avoidance and greater cynicism about advertising messages. HR and PS data were highly concordant in identifying differences between the three key HCW categories (doctors, nursing/allied-health, support services) and predicting appropriate implementation strategies. Among doctors, the data suggest that key messaging should differ between full-time vs part-time (visiting) senior medical officers (SMO, VMO) and junior hospital medical officers (HMO), with SMO messaging focused on evidence-based compliance, VMO initiatives emphasising structured mandatory controls and prestige loss for non-adherence, and for HMOs focusing on leadership opportunity and future career risk for non-adherence. DISCUSSION: Compared to current standardised approaches, targeted interventions based on personality differences between HCW categories should result in improved infection control-related culture-change uptake. Personality profiling based on HR data may represent a useful means of developing a national culture-change “blueprint” for HCW education.