Aspects of the work environment influence employee well-being. However, it is unclear how employee lifestyle behaviours, health characteristics and well-being may differ within a broader occupational sector. To investigate the health characteristics, lifestyle behaviours and well-being of three Fire and Rescue Service (FRS) occupational groups that differ in shift work and occupational demands: operational firefighters (FF), emergency control (EC) and administrative support (AS) workers. Data were obtained via an online survey using previously validated questionnaires to assess health characteristics, lifestyle behaviours and perceived well-being. Differences between groups were explored, controlling for confounding variables, using analysis of covariance (ANCOVA) methods. Effect sizes are reported where appropriate to demonstrate clinical significance. Four thousand five hundred and sixty-four FRS personnel volunteered, with 3333 (73%) completing the survey out of a total workforce of 60000 (8%). FF reported the lowest prevalence of chronic medical conditions (10%), compared with AS (21%) and EC (19%) workers. Total physical activity (PA) was 66% higher among FF compared with EC and AS workers. Components of sleep and self-rated health were independent predictors of well-being irrespective of FRS role. FF reported the highest levels of PA and highest perceptions of well-being, and the lowest prevalence of obesity and chronic medical conditions, compared with other FRS occupational groups. These findings may be used to inform FRS workplace intervention strategies.