Health-related quality of life (HRQoL) is being increasingly studied as an indicator of wellbeing. This study evaluated the HRQoL of nursing students and relationships between lifestyle behaviors including smoking, diet, alcohol intake and physical activity. A cross-sectional study was conducted in 2018 using the Short-Form 12V2 to measure the HRQoL of 475 students from both regional and metropolitan universities in Queensland, Australia. z-scores were aggregated into a Physical Composite Score (PCS) and a Mental Composite Score (MCS). Multivariate linear regression was used to explore the associations. Nursing students (94.5% female) had lower HRQoL scores relative to the general Australian population. Students enrolled at the regional university, with higher income; higher intakes of vitamin A, calcium, and iodine; and more physical activity had a higher Mental Composite Score, but those with health conditions and high intakes of meat, fat, carbohydrates, and sugar reported lower Physical Composite Score compared to their counterparts. Skipping breakfast, physical inactivity, and alcohol score were inversely associated with HRQoL. This study highlights the need for strategies to address the poor lifestyle and HRQoL in nursing students to support their physical and mental health.