Background: Dehumanization refers to the tendency of individuals or groups to attribute fewer human characteristics to other individuals or groups (referred to as hetero-dehumanization) or to themselves (referred to as self-dehumanization). This phenomenon currently seems to predominate in the medical and nursing professions. Indeed, healthcare environments facilitate latent forms of dehumanization due to their structure, organization, and inherent professional demands. This study aimed to investigate the association between hetero- or self-dehumanization and personality traits of healthcare professionals, as well as the possible key mediating role of empathy in this relationship. Methods: A total of 1150 healthcare employees were recruited for the current study with a mean age of 45.13 years. Data were collected through a questionnaire completed by health professionals. Results: Statistically significant relationships were found between self- and hetero-dehumanization and most personality traits (extraversion, agreeableness, conscientiousness). A mediation analysis revealed that empathy mediates the effects of personality traits on hetero- and self-dehumanization. Conclusions: The present study addresses the vital role of personality traits of healthcare professionals on dehumanizing oneself or patients, offering insights into improving therapeutic relationships through the cultivation of empathy.