The Work Ability Index (WAI) is the most commonly used tool for evaluating work capacity. Self-assessments made by workers can be influenced by various occupational and emotional factors. We wanted to study the association of work-related factors, such as work annoyance, stress, overcommitment, job satisfaction, social support, and emotional factors, such as anxiety, depression, and happiness, with work ability, in a sample of 490 healthcare workers from an Italian public health company. A principal component analysis indicated the presence of two components of the WAI questionnaire; the first expresses "subjectively estimated work ability" (SEWA), and the second refers to "ill-health-related work ability" (IHRWA). Using stepwise multiple hierarchical linear regression, we identified the factors that best predicted the total score on the WAI and on the two components. The total score was negatively predicted by anxiety, depression, a lack of happiness, low job satisfaction, overcommitment, and work annoyance. Age, being female, anxiety, and occupational stress were associated with a reduction in the IHRWA component score, while overcommitment, work annoyance, a lack of social support, depression, and a lack of happiness were negatively associated with the SEWA component. These results can help interpret those of epidemiological studies and provide guidance on ways to improve work ability.