Abstract Background Stigma towards people with mental disorders (MD) in healthcare workers (HCWs) can negatively influence the quality of care provided and hinder patients from accessing healthcare. The aim of this study was to analyze stigma and its determinants among physicians and nurses in Italy, as a basis for future strategies to reduce its extent. Methods A cross-sectional study was conducted from April to September 2023. A sample of 1053 physicians and nurses working in Italy answered an online 52-item questionnaire. Variables explored demographic data, work context, burnout symptoms, and personal or family history of MD. The Opening Minds Scale for Health Care Providers (OMS-HC) was used to assess the level of stigma (score from 20 to 100, higher scores represent higher levels). Descriptive analysis and multivariable linear regression were performed. Results Females were 69.5% and median age was 48 years (IQR=37-58). Physicians were 60.5%. A total of 20% of the sample worked in psychiatric settings. Burnout symptoms were reported by 23.5%. Results showed a median OMS-HC score of 49 (IQR=43-54). Positive associations with stigmatizing attitudes were found for being a nurse (p = 0.020) and being in burnout (p = 0.017), while working in a psychiatric setting (p < 0.001) and having personal or family history of MD (p < 0.001) had negative associations. Conclusions This research unveiled a notable amount of stigma among Italian HCWs towards individuals with MD. Since people with MD experience higher rates of disability and mortality, it has to be a public health priority to develop targeted strategies to reduce stigma in HCWs, as it discourages patients from accessing healthcare. Future interventions should be tailored for different professionals and dealing with HCWs burnout. Key messages • Future interventions should consider that healthcare workers in burnout and nurses tend to have more stigma toward patients with mental conditions. • Healthcare professionals working in psychiatric settings and having personal or family history of mental disorders showed lower stigma.