People with HIV continue to experience HIV stigma. Quantitative data on HIV stigma perpetrated by healthcare providers of hospitals providing HIV care in high-income countries are limited. The aim of this study is to investigate factors associated with HIV stigma in Dutch healthcare settings from the healthcare providers’ perspective. We conducted a cross-sectional study using the questionnaire ‘Measuring HIV Stigma and Discrimination Among Health Facility Staff – Monitoring Tool for Global Indicators’ to assess HIV stigma among healthcare providers (n = 405) in two academic hospitals. Healthcare providers licensed to provide medical care were eligible for inclusion. The primary outcome was the self-reported prevalence of at least one manifestation of HIV stigma measured by six stigma indicators (four individual, two institutional). Secondary outcomes were the prevalence of HIV stigma per indicator, per occupation, per department, and factors associated with individual stigma indicators. HIV stigma was prevalent among 88.1% (95%CI 84.5% − 91.2%) of participants. Stigma was mostly driven by negative attitudes towards people with HIV and worry to acquire HIV. Multivariate analysis showed that several factors were associated with HIV stigma, including younger age, male sex, working at one of the surgical departments, and working as a nurse. Having received any training on HIV stigma and/or discrimination was associated with less HIV stigma among all indicators. In conclusion, HIV stigma is highly prevalent among Dutch healthcare providers. Targeted approaches, including training on HIV stigma and discrimination, are needed to reduce HIV stigma in healthcare and should, among others, focus on younger healthcare providers.