Objectives: To explore if there are differences in shift patterns and work-related factors between metropolitan and regional/rural healthcare shift workers and their risk of poor sleep and mental health. Furthermore, explore whether these factors impact on medical errors, workplace and car/near car accidents. Design: A cross-sectional study. Setting: An anonymous online survey of healthcare shift workers in Australia. Participants: A total of 403 nurses, midwives and paramedics completed the survey. Main outcome measures: Sample characteristics, employment location, shift work-related features, sleep and mental health measurements, workplace accidents, medical errors and car/near car accident post shift. Results: Regional/rural healthcare shift workers were significantly older, had more years' experience, worked more nights, on-call and hours per week. Those in metropolitan areas took significantly longer (minutes) to travel to work, had higher levels of anxiety, increased risk of shift work disorder, reported significantly more workplace accidents and were more likely to have a car/near car accident when commuting home post shift. Both groups reported ~25% having a medical error in the past year. Workplace accidents were related to more on-call shifts and poor sleep quality. Medical errors were associated with fewer years' experience, more evening shifts and increased stress. Car accidents were associated with metropolitan location and increased depression. Conclusion: Differences in work-related factors between metropolitan and regional/rural healthcare shift workers were observed. Some of these factors contributed to occupational health and safety risks. Further exploration is needed to understand how to reduce occupational health and safety risks, and improve employee and patient safety both in both regional/rural and metropolitan areas.