While considerable research has documented stigma toward key populations affected by HIV and AIDS – men who have sex with men (MSM), sex workers (SWs) – it provided limited empirical evidence on the presence of layered stigma among health-care professionals providing services for these populations. C-Change conducted a survey among 332 staff of health-care and social service agencies in Jamaica and The Bahamas to understand the levels of stigma toward people living with HIV (PLHIV), including MSM and SWs and factors associated with stigma. While most health-care professionals responding to the survey said that PLHIV, MSM, and SWs deserved quality care, they expressed high levels of blame and negative judgments, especially toward MSM and SWs. Across a stigma assessment involving eight vignette characters, the highest levels of stigma were expressed toward PLHIV who were also MSM or SWs, followed by PLHIV, MSM, and SWs. Differences were assessed by gender, country, type of staff, type of agency, and exposure to relevant training. Findings indicate higher reported stigma among nonclinical vs. clinical staff, staff who worked in general vs. MSM/SW-friendly health facilities, and among untrained vs. training staff. This implies the need for targeted staff capacity strengthening as well as improved facility environments that are MSM/SW-friendly.