Workplace stress can negatively impact healthcare providers' professionalism and quality of care. One source of workplace stress is the experience of workplace mistreatment. Drawing on the Job Demands-Resources theory, this study aimed to (a) investigate the impact of mistreatment frequency experienced by healthcare workers from insider sources (i.e. co-workers, supervisors) and outsider sources (i.e. patients, visitors) on affective ill-being and (b) the potential moderating role of trait resilience and trait self-efficacy, as personal resources, in the mistreatment-ill-being relationship. Lagged design. We collected data from 153 Irish healthcare workers between January 2018 and June 2019 via three surveys, separated by 1-week intervals. Personal resources were measured at Time 1, frequency of mistreatment from the two sources was assessed at Time 2 and affective ill-being was assessed at Time 3. We used moderated regression analyses to evaluate the association of mistreatment frequency from the two sources and affective ill-being and the moderating effect of personal resources. Only insider mistreatment frequency was positively related to affective ill-being. Furthermore, the positive impact of insider mistreatment on affective ill-being was moderated by self-efficacy (but not by trait resilience). In contrast to our expectations, the relationship was stronger at high levels compared to low levels of self-efficacy. Mistreatment from co-workers and supervisors is linked to higher levels of affective ill-being. Additionally, healthcare workers with high self-efficacy were more vulnerable to the negative consequences of co-worker and supervisor mistreatment as it impacted their affective ill-being. These findings extend the literature on workplace mistreatment by integrating insider and outsider perpetrators of mistreatment and examining their differential impact on the employees' affective ill-being. It also highlights mistreatment from organizational insiders as a significant factor in the relationship between mistreatment and affective ill-being. No patient or public contribution. WHAT DOES THIS PAPER CONTRIBUTE TO THE WIDER GLOBAL COMMUNITY?: The findings underscore the detrimental impact of workplace mistreatment on the well-being of healthcare professionals. The study findings of higher frequency of insider mistreatment being associated with increased affective ill-being call for action, with line managers and supervisors being critical to achieving respective changes in healthcare workers' environment. The findings have implications for policymakers interested in developing a framework to support healthcare professionals in addressing workplace mistreatment, enabling them to effectively fulfil their role as care providers.