The predictive value of work ability for several health and occupational outcomes is well known. Maintaining the ability to work of all employees has become an important topic in research although some evidence suggests that some groups of workers need greater attention than others. Healthcare workers (x¯ = 54.46 ± 5.64 years) attending routine occupational health checkups completed their work ability, occupational risk and sociodemographic measures. An analysis examined whether work ability differed according to gender, age and professional category. Mediation of these relationships by occupational risk variables, such as work–family conflict, was examined. Females and older adults had worse work ability than their counterparts. Professional group was not independently associated. Gender-related differences were mediated by current and historic ergonomic risk, psychosocial risk and work–family conflict. Age-related differences were mediated by violence/discrimination at work. All job risk variables, apart from current ergonomic risk, mediated associations between professional category and work ability. The present study identified the importance of risk variables for the work ability of health workers according to gender, age and professional job type. Perceptions of work–family conflict and violence–discrimination seem particularly important and should be considered when targeting improvements in work ability.