Background Organisational culture is increasingly recognised as important for provision of high-quality long-term care. We undertook this study to measure organisational culture in residential aged care facilities in two Australian states. Methodology/Principal Findings Cross-sectional observational study in 21 residential aged care facilities in Western Australia (n = 14) and Queensland (n = 7), Australia. Staff and next-of-kin of residents participated. Measurement comprised surveys of facility staff and residents' next-of-kin, and structured observation of indicators of care quality. Staff tended to rate organisational culture positively. Some qualitative feedback from staff emphasised negative perceptions of communication, leadership and teamwork. Staffing levels were perceived as a dominant challenge, threatening care quality. Direct observation revealed variability within and between facilities but suggested that most facilities (n = 12) were in the typical range, or were quality facilities (n = 8). Conclusion There was scope to strengthen organisational culture in participating aged care facilities.